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Role of epidermal growth factor in human parturition: Betamimetic modulation of epidermal growth factor action in human amnion-derived cells
Su, Hsing-Chih, Ph.D.
The Ohio State University, 1991
UMI 300 N. Zeeb Rd. Ann Arbor, MI 48106 ROLE OF EPIDERMAL GROWTH FACTOR IN HUMAN PARTURITION:
BETAMIMETIC MODULATION OF EPIDERMAL GROWTH FACTOR
ACTION IN HUMAN AMNION-DERIVED CELLS
DISSERTATION
Presented in Partial Fulfillment of the Requirements for
the Degree Doctor of Philosophy in the Graduate
School of the Ohio State University
By
Hsing-Chih Su, B.S. *****
The Ohio State University
1991
Dissertation Committee: Approved by
Richard Fertel _ t . Douglas Kniss m g w g u A r£ \)lL ^ ______Sarah Tjioe Adviser John Enyeart Department of Pharmacology To My Husband And Parents ACKNOWLEDGEMENTS
I express my sincere appreciation to my advisor, Dr. Richard Fertel,
for his guidance and instruction throughout my graduate study. His
financial support has been greatly appreciated. Gratitude is expressed to Dr.
Douglas Kniss, for his assistance throughout this research project. Thanks
go to the other members of my dissertation committee, Drs. Sarah Tjioe,
John Enyeart and Richard Sams, for their suggestions and comments. I would like to thank my friends in Dr. Ferters laboratory, Dan Mullet and
Drs. George Cox, Jason Chang and Jeff Travers, for their friendship and
encouragement. I would also like to acknowledge other friends in Dr. Kniss'
laboratory, Eleanor Diss, Peter Zimmerman, Sandy Lohmier, Beth Kennard,
Anne Robinson, and Michael Waxman, for the help and enjoyable time
together. VITA
August 5, 1961 Bom - Taipei, Taiwan, Republic of China
1984 B.S., Taipei Medical College, Taipei, Taiwan, Republic of China Major: Pharmacy
1984-1985 Research Assistant, Taipei Medical College Department of Pharmaceutical and Medicinal Chemistry, Taipei, Taiwan, Republic of China
1986-present Graduate student, The Ohio State University Department of Pharmacology, College of Medicine Columbus, Ohio
PUBLICATIONS
Wang Y.-Z., Cooke HJ, Su H.-C., Fertel RH. Histamine augments colonic secretion in guinea pig distal colon. Am J Physiol 1990;258:G432-G439.
Kniss DA, Mershon J, Su H.-C., Sonek J, Fertel RH, Waxman M, lams JD, Gabbe SG. Evidence of a role for protein kinase C in epidermal growth factor-induced prostaglandin E2 synthesis in amnion cells. Am J Obstet Gynecol 1990;163:1883-1890.
Kniss DA, Mershon J, Sonek J, Su H.-C., Wu E, Gabbe SG. Protein kinase C augments growth factor-mediated prostanoid synthesis in cultured human amnion cells. 37th Annual Meeting of the Society for Gynecologic Investigation, March 21-24,1990, Poster number:511.
Su H.-C., Kniss DA, Fertel RH. Preexposure of WISH cells to endogenous factors augments epidermal growth factor-induced prostaglandin E2 synthesis. FASEB J 1990;4:A235. Su H.-C., Kniss DA, Fertel RH. Catecholamines alter EGF-induced PGE2 production in WISH cells via a cAMP-dependent pathway. FASEB J 1991; 5-.A392.
FIELDS OF STUDY
Major Field: Pharmacology
Studies in Biochemical Pharmacology
Pharmacology Department of Pharmacology faculty
Physiology Drs. Curry J., Bouiant J., Lipsky J., Kanabus E., and Pieper H.
Biochemistry Drs. Merola J., Brierley G., Sprecher H., Richardson K., Horrocks L., and Schumm D.
Radioisotopes Drs. Feller D., Malspeies L., and Brueggemeier R.
Neurochemistry Dr. Horrocks L.
v TABLE OF CONTENTS
Page DEDICATION ...... ii
ACKNOWLEDGMENT ...... iii
VITA ...... iv
LIST OF TABLES ...... x
LIST OF FIGURES ...... xi
INTRODUCTION ...... 1
I. Human parturition ...... 2
A. Definition of human parturition ...... 2
B. Physiological and biochemical events of human parturition ...... 2
II. Factors which initiate and control human parturition ...... 5
A. Progesterone ...... 5
B. Oxytocin ...... 6
C. Factors secreted from the fetus ...... 7
III. Role of prostaglandins in human parturition...... 9
A. Evidence for role of prostaglandins in human parturition ...... 10
B. Biosynthesis of prostaglandins ...... 10
vi C. Sources of uterine prostaglandins ...... 11
D. Mechanisms by which prostaglandins induce parturition ...... 17
IV. Role of epidermal growth factor in human parturition ...... 17
A. EGF as a candidate for the labor-initiating signal in hum ans ...... 18
B. Biochemical effects of EGF and its receptor ...... 18
C. Mechanisms by which EGF induces PGE2 production ...... 19
V. Preterm labor as an abnormality of parturition ...... 19
A. Infection-induced preterm lab o r...... 20
B. Pharmacologic control of preterm labor: Tocolysis...... 22
VI. Hypothesis...... 23
METHODS ...... 24
I. Cell culture techniques...... 24
A. Materials ...... 24
B. M ethods ...... 25
II. Biochemical te c h n iq u e s ...... 27
A. Protein assay ...... 27
B. Prostaglandin E2 radioimmunoassay ...... 29
C. Cyclic AMP radioimmunoassay...... 31
D. Radioreceptor assay...... 34
E. Incorporation of [3H]arachidonic acid into phospholipids ...... 36
vii F. Assay of PGH2 synthase activity ...... 38
RESULTS ...... 41
I. Effect of EGF on PGE2 production by WISH cells ...... 41
II. Effect of endogenous substances on EGF-induced PGE2 production ...... 44
III. Effect of catecholamines on EGF-induced PGE2 production ...... 48
IV. Role of cAMP in the inhibitory effect of catecholamines on EGF-induced PGE2 production ...... 51
V. Effect of tocolytic drugs on EGF response ...... 66
VI. Relationship between cAMP and PGE2 in WISH cells ...... 83
VII. Effect of catecholamines on EGF radioligand binding ...... 88
VIII. Incorporation of arachidonic acid into phospholipids ...... 99
IX. The activity of PGH2 synthase in WISH cells ...... 104
DISCUSSION ...... 109
I. Use of human amnion-derived WISH cells as a model system to study the regulation of PGE2 biosynthesis in parturition ...... 109
II. Role of EGF and catecholamines in human parturition ...... 110
III. Relationship between cAMP and PGE2 in WISH cells ...... 112
IV. Role of protein kinase C in EGF-induced PGE2 production ...... 116
V. Regulation of EGF receptors...... 117
VI. Regulation of PGH2 synthase activity ...... 118
vm• • • VII. Role of cytokines in preterm labor ...... 118
VIII. Conclusions and significance ...... 119
IX. Future studies ...... 121
LIST OF REFERENCES...... 123
ix LIST OF TABLES
TABLE Page
1. Factors associated with preterm labor ...... 21
2. Effect of propranolol on epinephrine-stimulated cAMP accumulation in WISH cells ...... 61
3. Effect of epinephrine and dibutyryl cAMP on EGF-induced PGE2 production by WISH cells ...... 89
4. Saturation binding isotherm of [125I]EGF binding to WISH cells pretreated with epinephrine ...... 98
5. Effect of epinephrine on I3H]arachidonic acid incorporation into phospholipids in response to EGF ...... 105
x LIST OF FIGURES
FIGURE Page
1. Cellular mechanisms controlling myometrial contractility .... 4
2. The organ communication system hypothesis of parturition ... 8
3. Proposed pathways for the mobilization of arachidonic acid from phosphatidylethanolamine and phosphatidyl- in o s ito l...... 12
4. The cyclooxygenase cascade pathway of arachidonic acid m e ta b o lism ...... 13
5. Interactions between the synthesis, transfer and metabolism of prostaglandins in the human fetal membranes and decidua ...... 16
6. Time-dependent effect of EGF on PGE2 production by WISH c e l l s ...... 42
7. Dose-dependent effect of EGF on PGE2 production by WISH c e l l s ...... 43
8. Lack of effect of oxytocin on EGF-induced PGE2 production ...... 45
9. Effect of IL-la on EGF-induced PGE2 production...... 46
10. Effect of epinephrine on EGF-induced PGE2 production 47
11. Time-dependent effect of pretreatment of WISH cells with epinephrine on EGF-induced PGE2 production ...... 49
xi 12. Dose-dependent effect of epinephrine on EGF-induced PGE2 production ...... 52
13. Dose-dependent effect of norepinephrine on EGF-induced PGE2 production...... 53
14. Dose-dependent effect of dopamine on EGF-induced PGE2 production ...... 54
15. Time-dependent effect of epinephrine on cAMP accumulation in WISH cells ...... 56
16. Dose-dependent effect of epinephrine on cAMP accumulation in WISH cells ...... 57
17. Time-dependent effect of norepinephrine on cAMP accumulation in WISH cells ...... 58
18. Dose-dependent effect of norepinephrine on cAMP accumulation in WISH cells ...... 59
19. Effect of butoxamine on epinephrine-stimulated cAMP accumulation ...... 62
20. Effect of propranolol on epinephrine-induced decrease in PGE2 production in response to EGF ...... 64
21. Effect of butoxamine on epinephrine-induced decrease in PGE2 production in response to EGF ...... 65
22. Effect of a cAMP analog on EGF-induced PGE2 production .... 67
23. Effect of an adenylate cyclase activator on EGF-induced PGE2 production ...... 68
24. Effect of a phosphodiesterase inhibitor on EGF-induced PGE2 production ...... 69
25. Time-dependent effect of forskolin on cAMP accumulation in WISH cells ...... 70
26. Dose-dependent effect of forskolin on cAMP accumulation in WISH cells ...... 71
xii 27. Dose-dependent effect of forskolin on EGF-induced PGE2 production ...... 72
28. Dose-dependent effect of terbutaline on EGF-induced PGE2 production ...... 74
29. Effect of ritodrine on EGF-induced PGE2 production ...... 75
30. Time-dependent effect of terbutaline on cAMP accumulation in WISH cells ...... 77
31. Time-dependent effect of ritodrine on cAMP accumulation in WISH cells ...... 78
32. Dose-dependent effect of terbutaline on cAMP accumulation in WISH cells ...... 79
33. Dose-dependent effect of ritodrine on cAMP accumulation in WISH cells ...... 80
34. Effect of butoxamine on terbutaline-induced decrease in PGE2 production in response to EGF ...... 81
35. Effect of butoxamine on ritodrine-induced decrease in PGE2 production in response to EGF ...... 82
36. Correlation between the effects of cAMP-stimulating agents on cAMP accumulation and on EGF-induced PGE2 production in WISH cells ...... 84
37. Time-dependent effect of PGE2 on cAMP accumulation in WISH cells ...... 85
38. Dose-dependent effect of PGE2 on cAMP accumulation in WISH cells ...... 86
39. Effect of EGF on cAMP accumulation in WISH cells ...... 87
40. Kinetics of [125I]EGF binding to WISH cells at 4 ° C ...... 90
41. Inhibition of [125I]EGF binding to WISH cells by EGF ...... 92
xiii 42. Effects of epinephrine and PMA on [125I1EGF competitive b i n d in g ...... 93
43. Effect of epinephrine on I125I]EGF specific binding...... 94
44. Saturation binding isotherm of [125I]EGF binding to WISH cells ...... 96
45. Rectangular hyperbola plot of [125I]EGF specific binding to WISH cells ...... 97
46. Time-dependent effect of [3Hlarachidonic acid incorporation into total phospholipids in WISH cells ...... 101
47. Incorporation of I3H]arachidonic acid into WISH cell phospholipids...... 102
48. Effect of EGF on [3H]arachidonic acid incorporation into WISH cell phospholipids ...... 103
49. Effects of epinephrine and EGF on PGH2 synthase a c tiv ity ...... 107
50. Effect of epinephrine on EGF-induced PGH2 synthase a c tiv ity ...... 108
51. Regulation of PGE2 production in amnion-derived WISH cells ...... 114
xiv INTRODUCTION
In 1977, Thorbum et al. stated: "We now recognize that in late pregnancy a train of events is initiated that ultimately results in the delivery of the fetus. However, we still do not know exactly how and where the train starts, or exactly how it exerts its ultimate action on the myometrial cell" [11. Despite many workers' efforts during the past decade, there is still no unified theory to explain human parturition - the process of childbirth.
Recent hypotheses have focused on the relationship between the fetus and the fetal membranes as a controlling factor in parturition. It has been proposed that substance(s) produced by the fetus may cause changes in the amnion and that this in turn initiates parturition [21. Several lines of evidence derived from studies in different species indicate that prostaglandins (PGs) are involved both in parturition and the softening and dilation of the cervix which immediately precedes parturition [3,41. Since preterm labor, a major cause of perinatal mortality and morbidity, shares a common terminal pathway (uterine contraction and cervical dilation [21) with term labor, and may be initiated by an interaction between infection and the amnion, causing an increase in PG synthesis [51, an understanding of the
1 2 mechanisms of human parturition may be helpful for management of preterm labor.
I. Human parturition
A. Definition of human parturition
Labor in human beings is defined as the entire process leading to the delivery of the infant and placenta [61. Labor is full term when it occurs after a period of gestation which is 40 weeks from the first day of the last menstrual period. Labor is usually divided into three stages [7,8]. The first stage begins with the onset of uterine contractions and ends at full cervical dilation. In the second stage of labor, the fetus completes its descent through the pelvis and is expelled from the uterus. The third stage begins at the delivery of the infant and ends with the delivery of the placenta.
B. Physiological and biochemical events of human parturition
In a normal pregnancy, the uterine cervix remains safely closed for the duration of pregnancy but can be opened with the physiological forces of labor. The process of cervical preparation for labor is called ripening, which is characterized by softening, effacement and dilation of the cervix [7,91.
The increase in water content together with the decrease in collagen concentration in cervical tissue may contribute to the softening of the cervix in preparation for labor [101. At the same time, the shape of the cervix 3 changes from a narrow, fibrous tube to a wide, dilated, soft canal. This
shortening of the cervical canals is called "effacement" and is normally
completed before the beginning of cervical dilation .
The onset of labor is defined by the spontaneous contraction of the myometrium, or uterine muscle. As with all muscle, myometrial contraction
is initiated by action potentials, which can be spread by cell to cell contacts
called "gap junctions" [51. The coupling of the action potential to a mechanical contraction is achieved by an increase in intracellular calcium
from 10'7 M to 10-6 M. This may occur from two sources. Calcium can enter the cell from the extracellular fluid through the plasma membrane via
calcium channels or be released from membrane-bound intracellular storage
areas. Contraction is terminated by a decrease in the intracellular calcium
concentration by way of transporting calcium either through the plasma membrane to the interstitial fluid or crossing the membranes of the
sarcoplasmic reticulum for intracellular storage.
A generally-accepted theory of myometrial contractility is that
calcium, in combination with calmodulin, activates myosin kinase which
phosphorylates the light chain of myosin (Fig. 1). Phosphorylated myosin
interacts with actin and causes contraction. When calcium is removed,
dephosphorylation occurs and the muscle relaxes [12]. Myometrial cell
activity can also be modified by raising intracellular cAMP, which may lower intracellular calcium levels and inhibit the myosin light-chain kinase, 4
magnesium sulfate I 13-adrenergic agonists calcium antagonists
u-Adrenerglc Receptor Adenvl Cvc ase
Intracellular Calcium cAMP Stores ► ca Na+/C a E xchange Ca - CM Protein Protein Wa+ * Kinases Kinases (Active) (Inactive)
MLK/Active) MLK (Inactive) NaVK
Myometrial Myosin P-myosln Contraction
Figure 1. Cellular mechanisms controlling myometrial contractility
Calcium binds to calmodulin (CM) to form an active calcium- calmodulin complex (Ca2+-CM), which interacts with myosin light-chain kinase (MLK), in turn, phosphoiylates myosin, permitting interaction with actin and thereafter contraction of myometrium. When the calcium level is reduced, MLK is inactivated, phosphatase dephosphorylates myosin, and the muscle relaxes. Myometrial contractility can also be inhibited by cAMP. Cyclic AMP phosphorylates a protein kinase, which in turn phosphorylates MLK, decreasing its affinity for Ca2+-CM 111]. 5 thus causing relaxation of the myometrium [131.
II. Factors which initiate and control human parturition
Despite researchers' efforts during the past decade, the mechanisms which initiate human labor are still not defined. It has been proposed that factors secreted either from the mother or from the fetus initiate and control parturition. These include progesterone, oxytocin, fetal products, and prostaglandins.
A. Progesterone
The role of progesterone in human labor has been expored primarily because of its role in parturition in sheep, which is currently the most well- defined model for the study of the physiologic events of parturition. In the sheep, there is a sharp rise in the secretion of fetal cortisol prior to parturition. Cortisol then acts on the placenta, leading to an increase in the activity of steroid 17a-hydroxylase. The increase in this enzyme activity first decreases progesterone secretion and then increases estrogen secretion
[15,16]. After progesterone withdrawal and a singe in estrogen secretion in the pregnant ewe, cervical ripening begins and gap junctions develop between myometrial cells. However, there are differences between sheep parturition and human parturition. For example: (1) in humans, there is no clear-cut increase in the secretion of cortisol, and cortisol infused into the 6 fetus or the mother does not cause human labor [17]; (2) in humans, there is no demonstrable withdrawal of progesterone prior to the onset of labor
[17]; (3) in humans, there is no steroid 17a-hydroxylase activity in placental tissues [18]. Although there is no measurable decrease in the concentration of progesterone prior to the onset of parturition in human beings, there is evidence that some form of progesterone withdrawal has a significant role in human parturition. For example, progesterone withdrawal in women can be induced by the removal of the corpus luteum in early human pregnancy.
This is followed by spontaneous abortion [19]. It has been postulated that progesterone is essential for the maintenance of pregnancy by causing the inhibition of PG production [14].
B. Oxytocin
The ability of oxytocin to induce labor in pregnant women at or near term has led to a hypothesis that oxytocin released by the neurohypophysis has a primary role in the initiation and maintenance of labor [13]. Evidence for this includes the finding that there is a significant increase in the number of oxytocin receptors in myometrial tissue at the end of gestation
[20]. In addition, oxytocin acts on endometrial tissue to stimulate the release of PGs [21]. However, since blood levels of oxytocin do not increase before labor, and the amount and pattern of PG production during oxytocin- induced labor is different from that which occurs during spontaneous labor 7
[22], it is probable that oxytocin is important primarily in the late stages of labor (including delivery of the placenta and uterine contraction to reduce blood loss), and in the maternal response to suckling, but is not crucial for the initiation of labor [23,24].
C. Factors secreted from the fetus
To date, the most viable explanation for the initiation of parturition is that there is a fetal-maternal organ communication system, which transmits signals from the fetus to the mother [25]. There are two arms of this organ communication system: a paracrine arm and an endocrine arm.
Components of the paracrine system include protein hormones, growth hormones and growth factors, while components of the endocrine system include estrogen and progesterone originating from the placenta. Since either too long or too short a gestation period has no biological advantage to the mother but may have life-threatening consequences to the fetus, it is reasonable to propose that the fetus may play a role in determining both the duration of pregnancy and the onset of parturition [2]. That is, the communication between the fetus and the mother may occur by way of fetal secretory products that enter the amniotic fluid through fetal urine, skin, lungs, and mucosal surface (Fig. 2). 8
Amnlollc fluid Amnion Chorion Decidua Myomelrium
1 * Phospholipids labor-initiating - 4-1 1 Arachidonic acid signals -±LU^,
Figure 2. The organ communication system hypothesis of parturition
Fetal products (labor-initiating signals) excreted into the amniotic fluid are thought to regulate prostaglandin production within fetal membranes and decidua and thus influence the onset of labor [26]. Fetal products excreted into amniotic fluid are thought to regulate prostaglandin production within fetal membranes and decidua and thus influence the onset of labor. Both lipid and protein factors have been implicated. For example, Strickland et al [27] reported that a low molecular weight steroid or lipid in human urine stimulated PG synthesis in the bovine seminal vesicle, and Casey et al. [28] demonstrated a protein in amniotic fluid that increased PGE2 production by human amnion cells.
More recent studies indicate that Platelet-activating factor (PAF), which is excreted from the fetal kidney and I or fetal lung, accumulates as labor progresses [29,30], and increases PGE2 production by amnion tissue [31].
Epidermal growth factor (EGF), which may be a product of the fetal kidney
[32], is also present in human amniotic fluid during labor, and stimulates
PGE2 production in amnion cells [33].
III. Role of prostaglandins in human parturition
Although the mechanisms of the initiation and maintenance of human parturition are not defined, PGs are thought to play a critical role. The first evidence for a possible involvement of PGs in parturition was obtained in
1930 by Kurzrok and Lieb, who observed that an extract of human semen caused human myometrium strips to contract [34]. It has since been established that PGE2 and PGF2n stimulate uterine contractions at every stage of gestation. For this reason, many investigators assumed that endogenous 10 PGs are involved in the initiation of labor.
A. Evidence for role of prostaglandins in human parturition
Since the enzymes which synthesize and degrade PGs are not found in amniotic fluid [35], the level of PG in amniotic fluid is thought to be representive of PGs produced by the surrounding tissues and the fetus. In fact, PG levels in amniotic fluid correlate well with changes in the initiation and progress of labor. Several lines of evidence support a key role for PG in the onset of labor at term: (1) Labor is associated with increased concentrations of PGs in amniotic fluid [36,37]. Amniotic fluid levels of
PGE2, PGF2a and PGFM (15-keto-13,14-dihydro-PGF2a) have been found to be higher during labor than before its onset, and the concentrations of these
PGs increase as cervical dilation progresses; (2) PGs are able to induce abortion and labor [4]; (3) During spontaneous labor at term, levels of the
PG precursor arachidonic acid in amniotic fluid increase with progressive cervical dilation [38]; (4) Intraamniotic administration of arachidonic acid results in the onset of labor [39]; 5) PG synthetase inhibitors can delay the onset of labor at term [40,41] and preterm labor [42],
B. Biosynthesis of prostaglandins
The rate of PG formation appears to be regulated by the hydrolytic release of free arachidonic acid from esterified arachidonic acids [43]. 11 Arachidonic acid is stored mainly in phospholipids in the sn-2 position, from which it is liberated directly by phospholipase A2 or indirectly by the successive actions of phospholipase C, diacylglycerol lipase and monoacylglycerol lipase 144,451 (Fig. 3). The relative importance of these two routes in uterine tissue is uncertain but the data usually suggest that
phospholipase A2 is primarily responsible for the synthesis of PG associated with labor 1431. Arachidonic acid can be transformed into PG by the enzyme
cyclooxygenase [Fig. 41, which inserts two molecules of oxygen into
arachidonate to yield a 15-hydroperoxy-9,ll-endoperoxide with a substituted
cyclopentane ring (PGG2) [461. A hydroperoxidase reduces PGG2 to its 15- hydroxy analogue (PGH2). The PGH2 formed then isomerizes to the stable
substances PGE2, PGF2a, and PGD2 and is also transformed into two unstable products, PGI2 and thromboxane (TXA2). In a given tissue, the proportion
of each end product depends on the relative activities of the specific synthases in that tissue. For example, the most active synthase in amnion is PGE2 synthase, while in decidua the most active enzyme is PGF2a synthase
[43].
C. Sources of uterine prostaglandins
It has been demonstrated that neither biosynthesis nor degradation of
PGs occurs in amniotic fluid [351. However, placenta, myometrium, decidua vera, chorion laeve and amnion tissue all metabolize arachidonic acid and 12
Phosphatidylinositol Phosphatidylethanolamine
| (2)
Diacylglycerol (0
j(3) (4) Monoacylglycerol Arachidonic acid
Figure 3. Proposed pathways for the mobilization of arachidonic add from phosphatidylethanolamine and phosphatidylinositol
The reactions illustrated are catalyzed by (1) phospholipase A2, (2) phospholipase C, (3) diacylglycerol lipase, and(4) monoacylglycerol lipase. 13
Arachidonic acid
Cyclooxygenase
[PGG2 - » w i I ^ J
x j i pg i2 p g e 2 PGF2_alpha Thromboxane A2
6-Keto-PGFj_alpha Thromboxane B2
Figure 4. The cydooxygenase cascade pathway of arachidonic add m etabolism
Arachidonic acid is oxygenated by the enzyme prostaglandin synthetase or cydooxygenase to form PGG2/ which, is converted to PGH2. The PGH2 formed isomerizes to form a family of prostaglandins. 14 produce biologically active products [471. Therefore, PGs found in amniotic
fluid must be from neighboring tissues, e.g., uterine and intrauterine tissues.
(1) Prostaglandin synthesis in fetal membrane
It is clear that the fetal membranes, amnion and chorion, are important sources of PGs during labor [48,491. The fetal membranes are
contiguous with the amniotic fluid on one side and the decidua vera on the
other. Thus, fetal membranes are in direct communication both with the mother and with the fetus through secretions from the fetal lung, skin, or urinary and gastrointestinal tracts. It has been shown that fetal membranes are enriched with phosphatidylethanolamine containing arachidonic acid, the precursor of PGs of the 2-series. During labor, the arachidonic acid content of the fetal membranes is significantly decreased, suggesting mobilization and utilization [511. The activity of phospholipase A2 in fetal membranes increases as pregnancy progresses, particularly in the amnion. Furthermore, phospholipase A2 from fetal membranes has substrate specificity for phosphatidylethanolamine containing arachidonic acid in the sn-2 position
[52,531. These findings suggest that PG production is regulated through the control of phospholipase A2 activity in fetal membranes. 15 (2) Role of amnion in the initiation of human parturition
It has been found that at various stages of pregnancy, concentrations of arachidonic acid in amnion are several times higher than in chorion, suggesting an important role for amnion in the initiation of labor. Indeed, some investigators have suggested that the amnion is the major site of increased PG production at the time of labor [54].
Several characteristics of human amnion support the importance of this tissue in the initiation of parturition: 1) among intrauterine tissues,
amnion is the most active producer of PGs [55,47]; 2) PGs produced in
amnion are not metabolized in situ but can cross the membranes by
diffusion into the amniotic fluid and to neighboring tissues [55]; 3) amnion has a large surface area that is ideally suited for absorption and for responding to hormones and other substances in amniotic fluid; 4) amnion is contiguous with the chorion and close to the decidua vera, both of which produce PGs that can stimulate contractions in the myometrium.
(3) Transport of prostaglandins from fetal membrane to
m yom etrium
It is generally accepted that PGE2 is the major PG produced by the amnion, and that amnion has little capacity for PG catabolism [55].
PGE2 produced by human amnion at term may escape metabolism in the
chorion laeve and reach the decidua (Fig. 5). Indeed, approximately 70% of Amniotic fluid Amnion Chorion j Decidua Myometrium
PGE2 synthesis
elevated [PGE2 ]
9-Keto- reductase elevated [PGF 2-alpha ]
PGF 2-alpha synthesis
Figure 5. Interactions between the synthesis, transfer and metabolism of prostaglandins in the human fetal membranes and decidua
PGE2 produced by amnion may escape metabolism in the chorion and reach the decidua. In decidua, PGEj may be converted to PGF^ through 9- ketoreductase. PGEj and PGF2a may then reach the myometrium to cause contractile activity [56]. 17 PGE2 is unchanged after crossing through the chorion and decidua [57], In the decidua, PGE2 originating from the amnion and chorion laeve may be
converted to PGF2o( through 9-ketoreductase activity [58,591. PGE2 and PGF2„
from the decidua may then reach the myometrium to produce contractile
activity.
D. Mechanisms by which prostaglandins induce parturition
PGs induce parturition both by causing cervical ripening and
stimulating uterine contractility. It has been shown that PGE2 may act by
increasing the collagenolytic activity of cervical tissue to cause cervical ripening [3,60,61]. PGs also inhibit calcium uptake by the sarcoplasmic reticulum, increasing intracellular calcium levels and thereby stimulating myometrium activity [62]. In addition, PGs may modulate the size and number of gap junctions in the myometrium to facilitate contraction [63],
IV. Role of epidermal growth factor in human parturition
Epidermal growth factor (EGF) is a heat and acid-stable polypeptide with a molecular weight of 6045 [64,65]. EGF was first isolated by Cohen
[66,67] from the submaxillary gland of mice (mEGF) and since then, it has been identified in guinea pigs (g-pEGF), rats (rEGF), and human prostate
and urine (hEGF). It binds to specific receptors and causes a number of
biological responses in target cells, including mitogenesis and the 18 phosphorylation of membrane proteins in a variety of cell types [68,69].
A. EGF as a candidate for the labor-initiating signal in humans
EGF, which can be produced by the fetal kidney [32], may be the critical labor-inducing signal from the fetus to the myometrium. Evidence that EGF plays a critical role in human labor is based on the observation that concentrations of EGF in amniotic fluid rise with increased gestational age, and that the median concentration of EGF in amniotic fluid from women in labor is significantly higher than that in a control group of non-laboring women [70]. The importance of EGF in labor is further supported by the observation that amnion cells contain EGF receptors [71] and EGF stimulates
PGE2 production by amnion cells [72], Taken together, these results suggest that EGF plays a primary role as a fetal signal responsible for the initiation of early parturition events.
B. Biochemical effects of EGF and its receptor
The ability of EGF to stimulate PGE2 production in human amnion is mediated via the EGF receptor, since an anti-EGF receptor monoclonal antibody blocks the stimulatory effect [73]. The EGF receptor is a 170 Kd transmembrane glycoprotein with four functional domains [74]: an N- terminal, extracellular, EGF binding domain; a transmembrane domain; a cytoplasmic domain containing the tyrosine kinase activity; and a C-terminal 19 domain containing EGF-stimulated autophosphorylation sites. EGF binding to the extracellular receptor domain stimulates receptor tyrosine kinase activity and leads to increased intracellular phosphorylation and self-phosphorylation [74].
C. Mechanisms by which EGF induces PGEZ production
PGE2 production by amnion cells is controlled both by the release of arachidonic acid from cell phospholipids, and the conversion of arachidonic acid to PGE2. Arachidonic acid release could be stimulated by EGF in one or more of the following ways: by indirect stimulation of PLA2 as a result of an EGF-mediated increase in intracellular concentrations of calcium; by stimulation of PLC with activation of PLA2 consequent to the IP3-induced increase in cytosolic calcium levels; or by direct stimulation of PLA2.
Arachidonic acid conversion to PGE2 could be stimulated by EGF by an increase in synthesis of PGH2 synthase [71].
V. Preterm labor as an abnormality of parturition
Labor is considered preterm when it occurs in a pregnant woman whose period of gestation is less than 37 completed weeks from the first day of the last menstrual period. The incidence of preterm birth in the United
States ranges from 5 to 10 % [75-77] despite the remarkable success achieved in neonatal intensive care units. Preterm birth still accounts for at least 70% 20 of all perinatal deaths [78].
A. Infection-induced preterm labor
Preterm labor is a major cause of perinatal mortality and morbidity, but in many cases its etiology remains unknown [79,80]. Risk factors
associated with an increased incidence of preterm labor are listed in Table
1 [81]. Several studies have reported a strong association between preterm
labor and infection [2, 82-85]. It has been proposed that PG plays a role in
infection-induced preterm labor [86-89], and, in fact, amniotic fluid
concentrations of PGE2 and PGF2a are increased in women with preterm
labor and intraamniotic infection [88,89].
There are several possible sources for the increased PG in amniotic
fluid during infection. First, it has been demonstrated that microorganisms
have phospholipase activity which can cause an increase in arachidonic acid
production by the fetal membranes [90]. Second, microbial products have
also been demonstrated to stimulate PG production in intraamniotic tissues
[91]. For example, bacterial endotoxin can induce the production of tumor
necrosis factor (TNF) by the host cell. This may stimulate PG production by
intrauterine tissues. Finally, interleukin-1 (IL-1) and PAF are elevated in the
amniotic fluid of women with intraamniotic infection, and both can increase
PG biosynthesis by human amnion [31,92]. 21
Table 1. Factors associated with preterm labor
Maternal Factors Infectious Factors
Age (<17 or >35) Urinary tract infections Socioeconomic status Cervicitis vaginitis Marital status Premature membrane rupture Short stature Phospholipase release by Low prepregnancy weight local organisms First trimester bleeding Stress Environmental Factors Poor obstetric history Race Height above sea level Low level education Substance abuse Insufficient corpus luteum Strenuous physical activity Medical diseases Previous preterm birth Nutritional Factors W ork Poor pregnancy weight Fetal Factors Closely spaced pregnancies Weight loss efforts Multiple gestation Fad diets Congenital anomalies Significant malnutrition Genetic disorders Iatrogenic Factors Placental Factors Inappropriate inductions/repeat Placenta previa cesarean sections Circumvallate placenta Misjudged gestational age Marginal cord insertion
Uterocervical Factors
Uterine malformations Cervical incompetence Leiomyomata uteri 22 The initiation of human parturition in the presence of infection may be regulated by the mother and/or the fetus [21. Infection can trigger parturition through activation of the maternal monocyte/macrophage system, increasing the secretion of monokines (TNF, IL-1, PAF, etc.) by the fetus.
Preterm labor can therefore be viewed as an abnormality of parturition which occurs when the maternal and/or intrauterine environment is hostile and threatens the well-being of the host.
B. Pharmacologic control of preterm labor: To colysis
Preterm labor may be prevented by using agents that inhibit the synthesis or release of PGs (ethanol, PG inhibitors) or by suppress the contractile response of the myometrial cells (magnesium sulfate, betamimetics). Betamimetics (especially ritodrine and terbutaline) are the most widely used and best understood of the tocolytic drugs.
It has been proposed that the tocolytic effect of betamimetic drugs is at the level of the myometrium. Beta-adrenergic receptor agonists interact with muscle surface receptors and activate adenylate cyclase, catalyzing the formation of cAMP from ATP (Fig. 1). This increased intracellular cAMP level may enhance the activity of the Na+/K+ ATPase ion exchange pump, increasing the extrusion of Na+ from the cytoplasm [6]. The resulting increased Na+ gradient increases the Na+/Ca2+ exchange process. The net result of this is a decrease in free cytoplasmic calcium and an inhibition of 23 muscle contractility. Cyclic AMP may also phosphorylate a protein kinase, which in turn phosphorylates myosin light-chain kinase, decreasing its
affinity for the Ca2+-calmodulin complex. This results in a diminished
interaction between myosin and actin [6].
VI. Hypothesis
There is increasing evidence to support the concept that the signal for
the initiation of parturition emanates from the fetus [15,43] and targets
amnion cells. Since EGF causes a time- and dose-dependent increase in PGE2
production by amnion cells [93,94], it may be the signal for parturition, and
amnion-derived PGE2 may be its effector. However, the factors which
influence the impact of EGF on PGE2 production by amnion have not been
examined. This study used a human amnion cell line (WISH) to test the
following hypothesis: (1) EGF plays a critical role in human labor by
stimulating PGE2 production in amnion cells, and the stimulatory effect of
EGF can be modulated by substances found in amniotic fluid (e.g.
catecholamines); (2) the modulatory effects of catecholamines on EGF-
induced PGE2 production are via a cAMP-dependent pathway; (3) the
modulatory effects of catecholamines on EGF-induced PGE2 production are
due to changes in number and/or affinity of the EGF receptor ; and (4) the
modulatory effects of catecholamines on EGF-induced PGE2 production are
due to changes in the activity of PGH2 synthase. METHODS
I. Cell culture techniques
A. Materials
(1) Cell culture medium supplements
Newborn calf serum (NCS) and gentamicin were obtained from
GIBCO (Grand Island, NY). L-glutamine and sodium pyruvate were
obtained from Sigma Chemical Co. (St. Louis, MO). A stock solution of 200 mM L-glutamine and 100 mM sodium pyruvate was prepared in distilled water and sterilized.
(2) Complete cell culture medium
The complete cell culture medium (F12/DMEM/NCS) consisted of a mixture of Ham's F-12 and Dulbecco's modified Eagle's medium
(F12/DMEM, 1:1, v/v) (GIBCO, Grand Island, NY) adjusted to pH 7.2 and
supplemented with 10% NCS, 2 mM L-glutamine, 1 mM sodium pyruvate
and 50 u g lm lgentamicin.
24 25 (3) F10/HEPES/FAF-BSA medium
F-10 medium powder was obtained from GIBCO, Grand Island, NY
and was prepared at pH 7.2 in distilled water with 15 mM N-2-hydroxyethyl-
piperazine-N'-2-ethanesulfonic acid (HEPES) and 1 mg/ml fatty acid free- bovine serum albumin (FAF-BSA).
(4) Balanced salt solutions
Phosphate buffered saline (PBS) was prepared at pH 7.2 in distilled water as follows:
NaCl 8.0 g/L KC1 0.2 g/L Na2HP04 1.15 g/L KH2P04 0.2 g/L
Hank's balanced salt solution (HBSS) was obtained as 10 X stock
solution from GIBCO, Grand Island, NY. and was prepared at pH 7.2 in
distilled water with 15 mM HEPES and 1 mg/ml FAF-BSA.
B. M ethods
(1) Culture and maintenance of cells
The WISH cell line derived from amnion tissue was obtained from the
American Type Culture Collection (ATCC CCL25). The cell line was
established from altered colonies appearing in a subculture of primary
monolayer of amnion cells after a total of 35 days in vitro [50]. The cells 26 were cultured in a mixture of Ham's F-12 and Dulbecco's modified Eagle's medium (F12/DMEM, 1:1, v/v) with the addition of 10% newborn calf serum
(NCS), 2 mM L-glutamine, 1 mM sodium pyruvate and 50 itg/ml gentamicin, and were incubated at 37 °C in 5% COz/95% air. The growth medium was changed every two days. Upon reaching confluence, the cell cultures were subcultured at lower density for use. The cells were rinsed with HBSS once and removed from flasks by trypsin for 15 min at 37 °C in 5% COz/95% air.
The cells were collected into sterile 50 ml conical tubes and the trypsin was inactivated by addition of F12/DMEM/NCS. The pooled cell suspensions were centrifuged at 100 x g for 5 min and then resuspended in
F12/DMEM/NCS. An aliquot of the cell suspension was counted in a hemocytometer and viability of the subcultured cells was assessed by trypan blue. Cells were then seeded into appropriate flasks to maintain the cell line or plated in appropriate culture dishes for experiments.
(2) Counting and determination of cell viability
Cells were counted using a hemacytometer. Trypan blue (0.04% tiypan blue solution in PBS) was used to determine cell viability. Usually,
0.1 ml of trypan blue solution was added to an equal volume of cell suspension and allowed to stand for 2 minutes before loading onto a hemacytometer. Cells on the four large comer squares from the top and bottom of the hemacytometer were counted. The following equations were 27 used to calculate the cell density and viability:
cell density (number of cells/ml) =
number of cells counted x dilution factor x 104 (1 ) number of squares counted
% viability =
(total cells - trypan blue-stained cells) x 100% (2) total cells
(3) Cryostorage of cells
Cryostorage was used to retain a viable stock of cells. Cells in log
growth phase were trypsinized, centrifuged, and resuspended in complete
.medium. One ml of the cell suspension ( approximately 6 x 106 viable
cells/ml) was placed in a 2-ml sterile cryogenic vial and 1 ml of 20%
dimethylsulfoxide (DMSO) was added to the vial drop by drop. The vials were then frozen at -70 °C freezer overnight. Thereafter, the vials were
stored in liquid nitrogen. When required, the vials were thawed rapidly and
cell were reseeded into flasks at high cell density.
II. Biochemical techniques
A. Protein assay
Cell protein was measured by the method of Lowry et al. [951. 28 (1) Reagents
Reagent A. 2% Na2C03 in 0.1 N NaOH
Reagent B. 0.5% CuS04 ' 5HzO in 1% Na/K tartrate
Reagent C. 50 parts of reagent A and 1 part of reagent B
Reagent D. Phenol reagent (Folin & Ciocalteau) was titrated against
NaOH and was adjusted to 2 N.
Bovine serum albumin (BSA) (Sigma Chemical Company, St. Louis,
MO) was used as the protein standard. One mg/ml stock solution was
aliquoted and stored at -20 °C in a freezer.
(2) Preparation of the standards and samples
Four hundred and eighty fil of the 1 mg/ml BSA stock was diluted
w ith 720 n 1 of 0.1 N NaOH to yield 80 /tg/200 pi. Using a serial 1:1 dilution with 0.1 N NaOH, a six-point standard curve done in duplicate was
prepared, including a NaOH blank which was used to zero the photometer.
Absorbance was linearly proportional to the amount of protein ranging from
5 to 80 ptg per tube. Samples were solubilized and diluted in 0.1 N NaOH
prior to assay.
(3) Procedure
Two hundred pi of each standard or sample was added to duplicate
12 mm x 75 mm glass tubes. Two ml of regent C was added, and the tubes 29 were vortexed and incubated for 10 minutes at room temperature. One hundred /t 1 of reagent D was added while vortexing, and the tubes were
incubated for an additional 30 minutes at room temperature. Absorbance at
580 mn was determined using a spectrophotometer.
B. Prostaglandin E2 radioimmunoassay
(1) Reagents
Tris buffer - 0.05 M Tris base in distilled water, pH 7.5, with 0.1%
BSA and 0.05% sodium azide
Charcoal solution - 0.25% Norit A charcoal and 0.025% dextran T 70
in Tris buffer
Antiserum - The PGE2 antiserum was developed in this laboratory [96]
by inoculating domestic chickens with PGE2-keyhole
limpet hemocyanin conjugate, and is highly specific
and sufficiently sensitive to measure 10 picomoles of
PGE2.
PGE2 standard - PGE2 obtained from The Upjohn Company
(Kalamazoo, Michigan) was used as PGE2 standard.
Radiolabelled PGE2 - [5,6,8,11,12,14,15 - 3H (N)] PGE2 was purchased
from Amersham (Arlington Hts., IL).
Scintillation cocktail - ScintiVerse E was obtained from The Ohio
State University Chemical Stores. 30 (2) Treatm ent of cells for R1A
Cells were seeded in 24-well culture plates (2 x 105 cells per well) with
F12/DMEM/NCS. Two days after plating, the cells were approximately 80-
90% confluent. The cells were then rinsed once with 1 ml of serum-free
F12/DMEM medium, and treated with test substance in 0.5 ml of
F12/DMEM/NCS medium in the indicated methods. At the end of treatment, the medium was collected and frozen at -70 °C prior to PGE2 RIA.
(3) Procedure
The PGEZ concentration in the culture medium was determined by
RIA. In brief, a twelve-point standard curve was constructed by pipetting a series of unlabelled PGE2 at concentrations ranging from 1.56 to 100,000 pg/100 pi or 100 pi of unknown to 10 mm x 75 mm glass tubes in duplicate.
One hundred pi of antiserum at a dilution to give approximately 35% binding, and 100 pi of [3H]PGE2 (approximate 10,000 cpm) were added to
each tube. Additional tubes were prepared for the determination of 100% binding (B„) and nonspecific binding (blank). B„ tubes contained 100 pi of
Tris buffer, 100 pi of antibody and 100 p\ of [3H]PGE2; blank tubes contained
200 pi of Tris buffer and 100 pi of [3H]PGE2; total tubes contained 700 pi of
Tris buffer and 100 pi of [3H]PGE2. The tubes were vortexed and incubated
at 4 °C for 16-18 hr. To remove the unbound PGE2, 0.5 m l of dextran-coated
charcoal suspension was added to the incubation tube, vortexed, and left for 31 10 m inutes at 4 °C. Thereafter, the tubes were centrifuged at 2000 x g for 20 minutes, and the supernatant was decanted into a scintillation vial and 2.5
ml of scintillation cocktail was added to the vial. Each vial was then read by a Beckman scintillation counter. The amount of PGE2 in the sample was
determined by comparison with the twelve-point standard curve. Statistical
significance was determined using the impaired, two-tailed Student's t-test.
Data were presented as mean ± SEM of six replicates per condition.
Equations used in the RIA analysis were as follows:
absolute binding = B0-blank I total-blank (3)
% Binding = sample-blank I B„ - blank (4)
C. Cyclic AMP radioimmunoassay
The cell culture samples were analyzed for cAMP by a modification of the m ethod of Steiner et al. [97].
(1) Reagents
Sodium acetate buffer - 0.05 M sodium acetate in distilled water, pH
6.28
Bovine gamma-globulin solution - 0.25% bovine gamma-globulin in
0.05 M sodium acetate buffer
BSA solution - 5% BSA in sodium acetate buffer
60% saturated ammonium sulfate - 780 g of ammonium sulfate in 2 L distilled water
Antisenun - Cyclic AMP antiserum was developed in this laboratory
and is highly specific and sufficiently sensitive to
measure 10 femtomoles of cAMP. The antiserum was
diluted in 0.05 M sodium acetate buffer with 5% BSA
to provide a trace binding of 25-35%.
Cyclic AMP standard - Adenosine 3': 5'-cyclic monophosphate sodium
salt was from Sigma Chemical Co. (St. Louis,
MO). The 0.1 fiM of stock solution was
prepared in sodium acetate buffer.
Radiolabelled cAMP - Adenosine 3': 5'-cyclic monophosphate (cAMP)
was radiolabelled with 12SI using a modification
of a method of Steiner et al.I97J. It was kindly
provided from the laboratory of Dr. Sue
O'Dorisio.
Acetylation reagent - A 2:5 (v/v) mixture of acetic anhydride and
triethylamine
(2) Treatm ent of cells for RIA
Cells were seeded in 12-well plates (5 x 10s cells per well) with
F12/DMEM/NCS. Two days after plating, the cells were approximately 80-
90% confluent. The cells were then rinsed once with 2 ml of 33 F10/HEPES/FAF-BSA medium, and treated with test substances in 1 ml of
F10/HEPES/FAF-BSA medium in the indicated methods. The reaction was stopped by the addition of 2 ml 10% trichloroacetic acid (TCA) into each well. The TCA solution was then collected and frozen at -70 °C prior to cAMP RIA.
(3) Procedure
The samples were centrifuged to remove the precipitated protein and the supernatant was extracted 3 times with a 1:3 (v/v) ratio of sample to water-saturated ethyl ether to remove trichloracetic acid (TCA). Any remaining ether was removed by a 10-min incubation in a 57° C water bath.
Cyclic AMP concentration was then measured by a modification of the radioimmunoassay procedure of Steiner et al [97]. In brief, a 16-point standard curve was constructed by a series of unlabeled cAMP at concentrations of 0.31 to 10,000 fmol/100 p i. One hundred pi of each standard or sample was added to duplicate 12 mm x 75 mm glass tubes. Ten pi of a 2:5 (v/v) mixture of acetic anhydride and triethylamine was added to each tube to increase the sensitivity of the assay. Fifty pi of [12SIJcAMP diluted in 0.25% bovine gamma globulin and 50 pi of anti-cAMP antiserum diluted in 0.05 M sodium acetate buffer (pH 6.28) containing 5% bovine serum albumin were added to each tube. Additional tubes were prepared for the determination of total binding (B0) and nonspecific binding (blank). 34
B„ tubes contained 100 fil of sodium acetate buffer, 50 /tl of antibody and 50
/tl of [125I]cAMP; blank tubes contained 150 /tl of sodium acetate buffer and
50 [d of [12SUcAMP; total tubes contained 50 /tl of [125I]cAMP. The tubes were then incubated at 4°C for 16-18 hr. To precipitate bound nucleotide, two ml of 60% saturated (NH4)2S04 solution was added. The samples were then centrifuged, supernatant removed and the remaining precipitate was
counted by a Beckman gamma counter. The amount of cAMP in the sample was determined by comparison with the sixteen-point standard curve.
Statistical significance was determined using the impaired, two-tailed
Student's t-test. Data were presented as mean ± SEM of six replicates per
condition. Equations used in the RIA analysis were as follows:
absolute binding = B0-blank I total-blank (3)
% Binding = sample-blank I B„-blank (4)
D. Radioreceptor assay
(1) Reagents
Binding medium - F10/HEPES/FAF-BSA medium
Unlabelled ligand - Epidermal growth factor was obtained from
Upstate Biotechnology, Inc. (Lake Placid, NY).
Radiolabelled ligand - 3-[12SUiodotyrosyl-human epidermal growth
factor ([125IJEGF) was obtained from
Amersham, Arlington Hts., IL. 35
Cell solubilizer - 1 N of NaOH
(2) Treatment of cells for radioreceptor assay
Cells seeded in either 24-well culture plates (2 x 10s cells per well) or
12-well culture plates (5 x 10s cells per well) were grown in F12/DMEM/NCS.
Two days after plating, the cells were rinsed once with serum-free
F12/DMEM medium. The cells were then treated with test substances in
either 0.5 ml (24-well plates) or 1 ml (12-well plates) of F12/DMEM/NCS
medium in the indicated methods. At the end of treatment, the cells were rinsed with F10/HEPES/FAF-BSA medium and cooled in F10/HEPES/BSA
m edium at 4 °C for 30 m in.
(3) Procedure
Competition studies. The cells were incubated with varied
concentrations of unlabeled EGF (0-250 ng/ml) at 4 °C for 15 min.
Thereafter, the cells were incubated with 50 pM of [12SI]EGF at 4 °C. After
4-hr incubation, the cells were rinsed three times with F10/HEPES/FAF-BSA medium, solubilized with 1 N of NaOH and counted in a Beckman gamma counter.
Saturation binding isotherms. The cells were incubated in the presence or absence of 250 ng/ml unlabeled EGF at 4 °C for 15 min. The
cells were then incubated with [125IJEGF ranging from 0.001 to 0.8 nM at 4 36 °C. After 4-hr incubation, the cells were rinsed three times with
F10/HEPES/FAF-BSA medium, solubilized with 1 N of NaOH and counted in a Beckman gamma counter.
Data analysis. Non-specific binding was determined in the presence of excess unlabeled EGF (250 ng/ml). Specific binding was then determined as the difference between total binding and non-specific binding. The data were plotted using the computer program InPlot (Graph-PAD Software, San
Diego, CA) on an IBM compatible computer and expressed as fmol of EGF bound per mg protein. A rectangular hyperbola plot was then used to determine the binding site concentration (Bmax) and apparent dissociation constant (Kd). The equation used to calculate Bmax and Kd is as followed:
Y = A * X I (B + X) (5)
A: Bmax (the same unit as Y)
B: Kd ( the same unit as X)
E. Incorporation of [3H] arachidonic acid into phospholipids
(1) Cell labelling
Cells were seeded in 60-mm dishes (2 x 106 cells per well) with
F12/DMEM/NCS. Two days after plating, the cells were incubated with the sodium salt of [5,6,8,9,11,12,13,14,15-N-3H1 arachidonic acid under the conditions described by Rittenhouse-Simmons 199]. In brief, a solution of
[3H]arachidonic acid in ethanol is dried under nitrogen. One drop of 6 N 37 NaOH and 2 ml of distilled water were added. The fatty acid salt was then mixed with F12/DMEM medium containing 0.5% FAF-BSA, and adjusted the pH to 7.2-7.4. The cells were then incubated in the [3H]arachidonic acid containing medium for the indicated times.
(2) Cell treatment
The cells were rinsed with F12/DMEM/0.5% FAF-BSA and treated with the indicated test substances. The reaction was terminated by adding
0.5 ml of methanol/HCl (100:6,v/v) to the dish. The dish was then stored at
-70 "Cina freezer prior to lipid extraction.
(3) Lipid extraction
Lipids were extracted by using a modification of the method of Folch et al. [100]. In brief, cells were scraped from the dish with a rubber policeman and transferred to a glass tube. The dish was then rinsed with an additional 0.5 ml of methanol and transferred to the corresponding tube.
The tube was then sonicated, and 2 ml of chloroform was added and the tube was vortexed. Thereafter, two ml of water was added and the mixture was centrifuged for 5 min at 2000 x g to enhance phase separation. The lower organic phase was transferred into a new tube and evaporated to dryness under a stream of nitrogen. The residue was then dissolved in a small amount of chloroform/ methanol (2:1 ,v/v). Aliquots of samples were 38 applied to silica plates for thin layer chromatography (TLC). A solvent system of chloroform/ethanol/ water/ triethylamine (30:34:8:35, v/v) was used as the mobile phase. After developing and drying, the TLC plates were exposed to iodine vapor to visualize standards. Identities of individual species were confirmed by comparison with simultaneously run authentic standards. Radioactive spots identified by comparison of the standards were scraped into scintillation vials and radioactivity was then determined by liquid scintillation spectroscopy.
F. Assay of PGH2 synthase activity
(1) Reagents
Sonication buffer: 50 mM of potassium phosphate buffer (pH 7.4)
containing 2 mM EDTA
Radiolabeled substrate: [5,6,8,9,11,12,13,14,1-N-3H] arachidonic acid
obtained from Amersham (Arlington Hts.,
IL.)
Co-factors: 4.2 mM of L-tryptophan, 5.1 mM of reduced glutathione,
and 3.5 n M of hematin
(2) Treatment of cells for assay
Cells were seeded in 60-mm dishes (2 x 106 cells per dish) with
F12/DMEM/NCS. Two days after plating, the cells were rinsed once with 39 serum-free F12/DMEM medium, and treated with the indicated test substances in 3 ml of F12/DMEM/NCS medium. At the end of treatment, the cells were scraped from the dishes and sonicated in potassium phosphate buffer (50 mM, pH 7.4) containing 2 mM EDTA. The sonicates were then centrifuged at 750 x g for 10 min, and supernatant was used as the enzyme source.
(3) Procedure
The conversion of arachidonic acid to PGE2 was determined by the modification of the method of Okazaki et al. [55]. Assay was conducted by incubation of aliquots of the supernatant with 2 jiCi of l3H]arachidonic acid
(specific activity: 211.1 Ci/mmol), 4.2 mM of L-tryptophan, 5.1 mM of reduced glutathione, and 3.5 /tM of hematin in 1 ml-total volume at 37 °C for
10 min. The reaction was terminated by the addition of 200 fil of 1 M acetic acid and 15 jig of non-labeled PGE2. The [3H]PGE2 was then extracted with
3 ml of ethyl acetate. The mixture was centrifuged for 5 min at 2000 x g to enhance phase separation. The upper organic phase was carefully transferred into a new tube and evaporated to dryness under a stream of nitrogen. The residue was then dissolved in a small amount of chloroform/methanol (2:1,v/v). Aliquots of samples were applied to silica plates for TLC. The upper layer of ethyl acetate/trimethylpentane/acetic acid/water (110:50:20:100, v/v/v/v) was used as the mobile phase. After 40 developing and drying, the TLC plates were exposed to iodine vapor to visualize standards. Identities of individual species were confirmed by comparison with simultaneously run authentic standards. Radioactive spots identified by comparison of the standards were scraped into scintillation vials and radioactivity was then determined by liquid scintillation spectroscopy. Data were presented as mean ± SEM of three replicates per condition. Statistical significance was determined using the unpaired, two- tailed Student's t-test. RESULTS
I. Effect of EGF on PGE2 production by WISH cells
Previous investigators have shown that EGF stimulates PGE2 production by primary cultures of amnion cells [931. In order to examine this phenomenon in the WISH cell system, the time and dose-dependence of the phenomenon were tested. A concentration of EGF (10 ng/ml), which had been previously shown to stimulate PGE2 production [93,94], was first added to cells for times from 30 min to 12 hr. The results (Fig. 6), indicate that PGE2 production by WISH cells began 1 hr after treatment with EGF and reached a maximum after 4 hr. The basal level of PGE2 in the absence of EGF remained low up to the end of the 12-hr incubation period. On the basis of this experiment, it was concluded that a peak response occurs at 4 hr. The cells were then incubated with various concentrations of EGF ranging from 0.78 to 50 ng/ml for 4 hr. As shown in Fig. 7, EGF at a concentration of approximately 10 ng/ml caused maximal stimulation of
PGE2 production. Half-maximal stimulation of PGE2 production was observed at approximately 3 ng/ml EGF. The stimulation decreased at EGF concentrations above 25 ng/ml. On the basis of these results, it was
41 42
.5 4 CL) -4-> 0 D- 3 CD e
1 2 ai LU 4 CD 1 CL
0 J______I______I______I______I______I______L 0 2 A 6 8 10 12 Time (hr)
Figure 6. Time-dependent effect of EGF on PGE2 production by WISH cells
Two days after plating, WISH cells were incubated in the presence (closed triangle) or absence (open triangle) of 10 ng/ml EGF in F12/DMEM/NCS medium. At the indicated time, the concentration of PGE2 in the medium was determined by RIA. The data represent mean ± SEM of six replicates per condition. 43
c • «—" t CD 3 O c_ CL ec n 2 c n
ai 1 LU CD □_
0 0 10 20 30 40 50 EGF (ng/ml)
Figure 7. Dose-dependent effect of EGF on PGE2 production by WISH cells
Two days after plating, WISH cells were incubated for 4 hr with the indicated concentrations of EGF in F12/DMEM/NCS medium. The concentration of PGE2 in the medium was determined by RIA. The data represent mean ± SEM of six replicates per condition. 44 concluded that exposure of WISH cells to EGF stimulates PGE2 production in a time- and dose-dependent manner, and further experiments were conducted at a concentration of 10 ng/ml of EGF and at least a 4-hr incubation period.
II. Effect of endogenous substances on EGF-induced PGE2 production
It is clear that at least one substance found in amniotic fluid, EGF, can stimulate PGE2 production. Since many other substances are present in amniotic fluid in normal or abnormal pregnancy, including oxytocin, catecholamines, and interleukin-1 (IL-1), it is reasonable to propose that the effect of EGF on PGE2 production may be modulated by other substances found in amniotic fluid. In order to test the hypothesis that preexposure of amnion cells to substances found in amniotic fluid alters EGF-induced PGE2 production, WISH cells were pretreated for 12 hr with either oxytocin, epinephrine, or IL-1. The cells were then exposed to EGF, and PGE2 production was measured. While preincubation with 10"6 M oxytocin had no effect on EGF-induced PGE2 production (Fig. 8), preincubation with 10 U/ml
IL-la caused a 750 % increase in EGF-PGE2 production (Fig. 9) and epinephrine (10 s M) caused a 75 % decrease in EGF-PGE2 production (Fig.
10). On the basis of these results, it is concluded that the stimulatory effect of EGF can be modulated by preexposure of WISH cells to substances present in amniotic fluid. 45
Posttreatment: [~1 Control cz • r—i 8 j^EGF 10 ng/ml CD -l-> O c_ CL 6 c n
c n c A
cu LU CD 2 CL
0 Control Oxytocin Pretreatment
Figure 8. Lack of effect of oxytocin on EGF-induced PGE2 production
Two days after plating, WISH cells were preincubated for 12 hr in the presence or absence of 10-6 M oxytocin in F12/DMEM/NCS medium. Thereafter, the cells were rinsed once with serum-free F12/DMEM medium and exposed to F12/DMEM/NCS medium in the presence or absence of 10 ng/ml EGF for an additional 10 hr. The concentration of PGE2 in the medium was determined by RIA. The data are presented as mean ± SEM of six replicates per condition. 46
25 c • r —t Posttreatment: CD 20 -4-) [~~1 Control O c_ EGF 10 ng/ml CL 15 CD
c n C 10
OJ LU CD 5 CL
0 Control IL-la Pretreatment
Figure 9. Effect of IL-la on EGF-induced PGE2 production
Two days after plating, WISH cells were preincubated for 12 hr in the presence or absence of 10 U/ml IL-la in F12/DMEM/NCS medium. Thereafter, the cells were rinsed once with serum-free F12/DMEM medium and exposed to F12/DMEM/NCS medium in the presence or absence of 10 ng/ml EGF for an additional 10 hr. The concentration of PGE2 in the medium was determined by RIA. The data are presented as mean ± SEM of six replicates per condition (***: p< 0.0001 compared with EGF treatment only). 47
Posttreatment: = 5 • r i CD [""Icontrol o 4 c_ ^EGF 10 ng/ml Q.
- t 3 c n — 2 ai LXJ CD I Q_ 1
0 Control Epinephrine Pretreatment
Figure 10. Effect of epinephrine on EGF-induced PGE2 production
Two days after plating, WISH cells were preincubated for 12 hr in the presence or absence of 10 s M epinephrine in F12/DMEM/NCS medium. Thereafter, the cells were rinsed once with serum-free F12/DMEM medium and exposed to F12/DMEM/NCS medium in the presence or absence of 10 ng/ml EGF for an additional 10 hr. The concentration of PGE2 in the medium was determined by RIA. The data are presented as mean ± SEM of six replicates per condition (*: p<0.05). 48 m. Effect of catecholamines on EGF-induced PGE2 production
Since it has been demonstrated that concentrations of catecholamines increase in amniotic fluid during late pregnancy [101,1021, and there are functional 0-adrenergic receptors in human amnion tissues [1031, it is reasonable to propose a role for catecholamines in amnion. The next series of experiments was designed to examine the time- and dose-dependence of the catecholamine effect.
Time-dependent effect of pretreatment on EGF-induced PGE2 production
The initial experiment (Fig. 10) showed an effect of 12-hr pretreatment with epinephrine on EGF-induced PGE2 production. In order to determine the minimal time required for pretreatment, WISH cells were preexposed to
10 s M epinephrine for times from 5 min to 4 hr. The results (Fig. 11) indicate that the cells required at least 4 hr of preexposure to epinephrine to cause a significant inhibition of EGF-induced PGE2 production. Based on these results and the initial findings that PGE2 production reached a maximum after 4-hr exposure to a concentration of 10 ng/ml EGF, subsequent experiments were designed using a concentration of 10 ng/ml
EGF, a 4-hr pretreatment with test substances and an additional 4-hr exposure to EGF. 49
6 c .r—i CD -4-> 5 O c_ CL 4 CD e c n 3 c
CVJ 2 LU CD Q_
0 0 1 2 3 A Time for pretreatment (hr)
Figure 11. Time-dependent effect of pretreatment of WISH cells with epinephrine on EGF-induced PGE2 production
Two days after plating, WISH cells were preincubated for the indicated times in the presence or absence of 10 s M epinephrine in F12/DMEM/NCS medium. Thereafter, the cells were rinsed once with serum-free F12/DMEM medium and exposed to F12/DMEM/NCS medium in the presence or absence of 10 ng/ml EGF for an additional 4 hr. The concentration of PGE2 in the medium was determined by RIA. The data are presented as mean ± SEM of six replicates per condition. (Open circle: control, medium only at both treatment phases; closed circle: pretreated with epinephrine only; open triangle: posttreated with EGF only; closed triangle: pretreated with epinephrine and posttreated with EGF; *: p<0.05.) 50 Dose-dependent effect of epinephrine on EGF-induced PGE2 production
The effect of epinephrine on EGF-induced PGE2 production in amnion
cells was further tested by incubating cells with varied concentrations of
epinephrine for 4 hr prior to an additional 4-hr exposure to EGF. The results
shown in Fig. 12 indicate that, while exposure to concentrations of 10'10 to
W 4 M of epinephrine alone had little effect on PGE2 production by WISH
cells, EGF-induced PGE2 production was inhibited in a dose-dependent
fashion by prior exposure of the cells to epinephrine. Epinephrine at
concentrations below 10'8 M caused no significant effect on the EGF
response. However, epinephrine at 10‘7,10"6,10'5, and 10"* M caused a 30.1%
(p = 0.0012), 40.6% (p = 0.0003), 50.4% (p< 0.0001) and 45.6% (p< 0.0001)
decrease in EGF-induced PGE2 production, respectively.
Effects of norepinephrine and dopamine on EGF-induced PGE2 production
Although it is clear that epinephrine can inhibit the EGF-induced
response, amniotic fluid contains other catecholamines, including
norepinephrine and dopamine. Experiments were designed to determine the
effect of these catecholamines on the EGF response. When cells were
preincubated with norepinephrine alone, at concentrations ranging from 10'7 to 1QA M, there was little effect on PGE2 production (Fig. 13). As with
epinephrine, prior exposure of the cells to norepinephrine caused a dose-
dependent decrease in EGF-induced PGE2 production. Norepinephrine at a 51 concentration of 10'7 M had no significant effect on EGF response. However, norepinephrine at 10"6, 10'5, and 10"4 M caused a 29.7% (p=0.0024), 25.2%
(p=0.0073), and 44.2% (p< 0.0001) decrease in EGF-induced PGE2 production, respectively. Like epinephrine and norepinephrine, dopamine alone had little effect on PGE2 production (Fig. 14) but inhibited EGF- induced PGE2 production. Dopamine at 10"6 M reduced by 24.8% (p=0.0107)
and at 10 s M reduced by 58.8% (p< 0.0001) EGF-induced PGE2 production.
On the basis of these experiments, it is concluded that preexposure of amnion-derived WISH cells to all tested catecholamines inhibits PGE2 production in response to EGF.
IV. Role of cAMP in the inhibitory effect of catecholamines on EGF-induced
PGE2 production
Since catecholamines increase the concentration of cAMP in target cells, and cAMP has been shown to influence PG synthesis in a number of tissues [104,106,118], it was of interest to determine whether the inhibitory effect of catecholamines on EGF-induced PGE2 production is dependent upon their ability to increase cAMP in the WISH cells. 52
cz 10 • H QJ A-> o 8 c_ a — 2 CL c n e 6 c n «XK c 4 OJ LU CD CL 2 A A
0 Control EGF 10 -9 -8 -7 -6 -5 -A treated Log [Epinephrine] (M) in pretreatment
Figure 12. Dose-dependent effect of epinephrine on EGF-induced PGE2 production
Two days after plating, WISH cells were preincubated for 4 hr with the indicated concentrations of epinephrine in F12/DMEM/NCS medium. Thereafter, the cells were rinsed once with serum-free F12/DMEM medium and exposed to F12/DMEM/NCS medium in the presence (closed triangle) or absence (open triangle) of 10 ng/ml EGF for an additional 4 hr. The concentration of PGE2 in the medium was determined by RIA. The data are presented as mean ± SEM of six replicates per condition (Control: cells received medium only at both treatment phases; EGF-treated: cells received medium at the pretreatment phase and 10 ng/ml EGF at the posttreatment phase. *: p<0.05, **: p< 0.001, ***: p< 0.0001 compared with EGF treatment only). 53
CD 3 A-> O c_ Q. CD e 2 c n
OJ LU 1 CD Q_
0 Control EGF-treated -7 6 5
Log [Norepinephrine] (M) in pretreatment
Figure 13. Dose-dependent effect of norepinephrine on EGF-induced PGE2 production
Two days after plating, WISH cells were preincubated for 4 hr with the indicated concentrations of norepinephrine in F12/DMEM/NCS medium. Thereafter, the cells were rinsed once with serum-free F12/DMEM medium and exposed to F12/DMEM/NCS medium in the presence (closed triangle) or absence (open triangle) of 10 ng/ml EGF for an additional 4 hr. The concentration of PGE2 in the medium was determined by RIA. The data are presented as mean ± SEM of six replicates per condition (Control: cells received medium only at both treatment phases; EGF-treated: cells received medium at the pretreatment phase and 10 ng/ml EGF at the posttreatment phase. *: p<0.05, ***: p< 0.0001 compared with EGF treatment only). 54
c r—f
o c_ a.
CD cz «KK C\J LU CD Q.
Control EGF-treated -8 7
Log [Dopamine] (M) in pretreatment
Figure 14. Dose-dependent effect of dopamine on EGF-induced PGE2 production
Two days after plating, WISH cells were preincubated for 4 hr with the indicated concentrations of dopamine in F12/DMEM/NCS medium. Thereafter, the cells were rinsed once with serum-free F12/DMEM medium and exposed to F12/DMEM/NCS medium in the presence (closed triangle) or absence (open triangle) of 10 ng/ml EGF for an additional 4 hr. The concentration of PGE2 in the medium was determined by RIA. The data are presented as mean ± SEM of six replicates per condition (Control: cells received medium only at both treatment phases; EGF-treated: cells received medium at the pretreatment phase and 10 ng/ml EGF at the posttreatment phase. *: p<0.05, ***: p < 0.0001 compared with EGF treatment only). 55 Time and dose-dependent effects of catecholamines on cAMF accumulation
in WISH cells
In order to test the hypothesis that the effect of catecholamines is the
result of their ability to increase cAMP in WISH cells, a concentration of Iff5
M epinephrine, which has previously been shown to stimulate cAMP in
amnion cells, was incubated with cells for times from 30 seconds to 30 min.
The results (Fig. 15) indicate that a 10-fold increase in cAMP accumulation
occurred within 30 seconds, followed by a rapid decline. Cyclic AMP was
essentially back to basal levels after 30 minutes. Although the response was
considerably diminished after 5 min, it was still clearly higher than controls.
For logistic purposes, all subsequent measurement of cAMP were made after
5 min of exposure to epinephrine. WISH cells were next exposed to
epinephrine at concentrations ranging from Iff9 to 10"4 M for 5 min. The
results (Fig. 16) indicate that epinephrine caused a dose-dependent increase
in cAMP accumulation in WISH cells. The amount of cAMP was not
significantly affected by epinephrine at concentrations of Iff8 M or less, and
reached a maximum at concentrations higher than 10-6 M. Similarly,
norepinephrine caused a transitory increase in cAMP accumulation (Fig. 17).
There was a rapid decline after exposure of cells to norepinephrine for 1
min. The peak response and the 5-min response were both lower than that
seen with epinephrine, probably because of the decreased /32-effect of
norepinephrine. Like epinephrine, norepinephrine caused a dose-dependent 56
CD 20 -t-J O c_ Q. 15 c n
o E 10 CL
Q_ 5 0 0 5 10 15 20 25 30 Time (min) Figure 15. Time-dependent effect of epinephrine on cAMP accumulation in WISH cells Two days after plating, WISH cells were incubated with 10-4 MIBMX in the presence (closed triangle) or absence (open triangle) of 10‘5 M epinephrine in F10/HEPES/BSA medium. At the indicated time, 2 ml of 10 % TCA was added to the well to stop cAMP metabolism. Cyclic AMP concentration was determined by RIA. The data represent the mean ± SEM of six replicates per condition. 57 5 c • r—1 CL) -»-> 4 O c_ CL CD 3 O e 2 CL 1 <=t C_) 0 Control -9 8 7 6 5 4 Log [Epinephrine] (M) Figure 16. Dose-dependent effect of epinephrine on cAMP accumulation in WISH cells Two days after plating, WISH cells were incubated with 5 x 10'5 M IBMX and the indicated concentrations of epinephrine in F10/HEPES/BSA medium. After a 5-min incubation, 10 % TCA was added to the plates to stop cAMP metabolism. Cyclic AMP concentration was determined by RIA. The data represent the mean ± SEM of six replicates per condition. 58 8 c • r—1 QJ -J-) O 6 c_ CL CD A O e CL 2 c_> 0 0 10 20 30 Time (min) Figure 17. Time-dependent effect of norepinephrine on cAMP accumulation in WISH cells Two days after plating, WISH cells were incubated with 10-1 MIBMX in the presence (closed triangle) or absence (open triangle) of 10 s M norepinephrine in F10/HEPES/BSA medium. At the indicated time, 2 ml of 10 % TCA was added to the well to stop cAMP metabolism. Cyclic AMP concentration was determined by RIA. The data represent the mean ± SEM of six replicates per condition. 59 CD 8 -*-> O '4 C_ CL 6 cn o e 4 CL 2 «=t U 0 0 10 20 30 40 50 Norepinephrine (^M) Figure 18. Dose-dependent effect of norepinephrine on cAMP accumulation in WISH cells Two days after plating, WISH cells were incubated with 10"4 MIBMX and the indicated concentrations of norepinephrine in F10/HEPES/BSA medium. After a 5-min incubation, 10 % TCA was added to the plates to stop cAMP metabolism. Cyclic AMP concentration was determined by RIA. The data represent the mean ± SEM of six replicates per condition. 60 increase in cAMP (Fig. 18). It should be noted that norepinephrine is clearly a less potent effector of cAMP than epinephrine in this system. On the basis of these experiments, it is concluded that both of the tested catecholamines stimulate cAMP accumulation in WISH cells in a time- and dose-dependent manner. Effect of /3-adrenergic antagonists on cAMP accumulation in response to epinephrine The greater effect of epinephrine on WISH cell cAMP suggested that the effect was mediated by a /3-receptor. In order to verify that the effect of epinephrine was mediated by the /3-adrenergic receptor, it was tested in the presence of the /3-blocker, propranolol. The stimulatory effect of epinephrine on cAMP accumulation was blocked by propranolol (Table 2). At a concentration of 10"6 M, propranolol was able to decrease epinephrine stimulation of cAMP by 65%, and 10‘5 M propranolol completely eliminated the epinephrine-stimulated increase in cAMP accumulation. Butoxamine, a selective /32-adrenoceptor antagonist, at a concentration of 10‘5 M also abolished epinephrine-stimulated cAMP accumulation (Fig. 19). From these experiments, it is clear that the epinephrine-induced increase in WISH cell cAMP occurs via the /32-adrenergic receptor. 61 Table 2. Effect of propranolol on epinephrine-stimulated cAMP accumulation in WISH cells Two days after plating, WISH cells were preincubated with 10"4 M IBMX in the presence or absence of either 10"6 M or 10 s M propranolol in F10/HEPES/BSA medium. After the preincubation phase, 10 s M epinephrine was added to some wells for an additional 5 min. Thereafter, 10% TCA was added to the plates to stop cAMP metabolism. Cyclic AMP concentration was determined by RIA. The data represent the mean ± SEM of six replicates per condition. Treatm ent______cAMP (pmol/mg protein) Control 1.85 ± 0.16 Propranolol (10-6 M) 1.83 ± 0.10 Propranolol (10‘5 M) 1.80 ± 0.20 Epinephrine (10’5 M) 8.08 ± 0.83 Propranolol (10-6 M) + Epinephrine 2.85 ± 0.50 Propranolol (10‘5 M) + Epinephrine 1.77 ± 0.13 62 8 c z • M CD A - > o 6 c_ CL c n 4 o s a Q. 2 0 C E B B+E Treatment Figure 19. Effect of butoxamine on epinephrine-stimulated cAMP accumulation Two days after plating, WISH cells were preincubated for 5 min with 10"4 M IBMX in the presence or absence of 10 s M butoxamine in F10/HEPES/BSA medium. After the preincubation phase, 1(T5M epinephrine was added to some wells for an additional 5 min. Thereafter, 10% TCA was added to the plates to stop cAMP metabolism. Cyclic AMP concentration was determined by RIA. The data represent the mean ± SEM of six replicates per condition. (C: cells received IBMX only; B: cells received IBMX and butoxamine; E: cells received IBMX and epinephrine; B + E: cells received IBMX, butoxamine, and epinephrine.) 63 Effect of /3-adrenergic antagonists on the epinephrine-induced decrease in EGF-induced PGE2 production If the inhibitory effect of epinephrine on PGE2 production induced by EGF is mediated by the /3-receptor, /3-blocking agents should decrease the inhibition. In order to test this, the effect of propranolol on the epinephrine-induced decrease in PGE2 production was determined. WISH cells were exposed to propranolol 30 min prior to epinephrine pretreatment. As shown in Fig. 20, propranolol at a concentration of 10 s M was able to block the inhibitory effect of epinephrine on EGF-induced PGE2 production. This is in accord with the previous result that propranolol at the same concentration eliminated the stimulatory effect of epinephrine on cAMP accumulation. Similarly, butoxamine at a concentration of 10‘5 M diminished the inhibitory effect of epinephrine on EGF-induced PGE2 production (Fig. 21). Since /32-adrenergic blockers abolished both the increase in cAMP accumulation and the inhibition of PGE2 production due to epinephrine, it is concluded that the inhibitory effect of epinephrine on EGF-induced PGE2 production may be mediated via an adenylate cyclase- coupled /32-adrenoceptor. « Effect of cAMP-stimulating agents on EGF-induced PGE2 production To confirm that the inhibitory effect of catecholamines on PGE2 production in response to EGF was via a cAMP-dependent pathway, WISH 64 a 5 • r*H CD -»-) o c_ A CL «x CD 3 CD C 2 ai LU CD CL 1 0 C EP P+E Pretreatment Figure 20. Effect of propranolol on epinephrine-induced decrease in PGE2 production in response to EGF Two days after plating, WISH cells were preincubated for 30 min with or without 10'5 M propranolol in F12/DMEM/NCS medium. Thereafter, 10‘5 M epinephrine was added to some wells for 3.5 hr. After the total 4-hr preincubation, cells were rinsed once with serum-free F12/DMEM medium and then exposed to 10 ng/ml EGF in F12/DMEM/NCS medium for an additional 4 hr. The concentration of PGE2 in the medium was determined by RIA. The data represent the mean ± SEM of six replicates per condition. (C: control; P: propranolol; E: epinephrine; P + E: propranolol and epinephrine. All four groups received 10 ng/ml EGF in posttreatment. **: p< 0.001 compared with control.) 65 6 5 CD -4-> O c_ 4 Q. CD 3 CD cr 2 CM LU CD Q_ 0 C Epi B Epi+B Pretreatment Figure 21. Effect of butoxamine on epinephrine-induced decrease in PGE2 production in response to EGF Two days after plating, WISH cells were preincubated for 30 min with or without 10'5 M butoxamine in F12/DMEM/NCS medium. Thereafter, 10’5 M epinephrine was added to some wells for 3.5 hr. After the total 4-hr preincubation, cells were rinsed once with serum-free F12/DMEM medium and then exposed to 10 ng/ml EGF in F12/DMEM/NCS medium for an additional 4 hr. The concentration of PGE2 in the medium was determined by RIA. The data represent the mean ± SEM of six replicates per condition. (C: control; B: butoxamine; E: epinephrine; B + E: butoxam ine and epinephrine. All four groups received 10 ng/ml EGF in posttreatment. ***: p< 0.0001 compared with control.) 66 cells were pretreated with either a cAMP analog (5 x 10 s M dibutyryl cAMP), an adenylate cyclase activator (5 x 10"6 M forskolin), or a 5-AMP phosphodiesterase inhibitor (5 x 10'5 M IBMX) for 4 hr prior to EGF exposure. Dibutyryl cAMP (Fig. 22), forskolin (Fig. 23) and IBMX (Fig. 24) all reduced PGE2 production in response to EGF. As with epinephrine, none of these three cAMP-stimulating agents alone had a significant effect on PGE2 production. As shown in Fig. 25 and Fig. 26, forskolin in the presence of IBMX stimulated cAMP accumulation in a time- and dose-dependent manner. It is probable that this increase in cAMP is correlated with the dose-dependent inhibition of EGF-induced PGE2 production which was observed (Fig. 27). On the basis of these experiments, it may be concluded that an increase in cAMP is directly correlated with a decrease in EGF-induced PGE2 production. V. Effect of tocolytic drugs on EGF response The most widely used tocolytic drugs are /?2-adrenergic agonists. Since previous studies indicated that the inhibition of EGF-induced PGE2 production is probably mediated by (tz receptors (Fig. 21), it was postulated that the clinically used tocolytic (32 agnoists, ritodrine and terbutaline, might also alter EGF-induced PGE2 production. 67 A Posttreatment: c r (~~| Control • r- 1 QJ -*-> ^ E G F 10 ng/ml O c_ 3 CL c n 2 c n c aj LJJ 1 CD Q_ 0 Control Db-cAMP Pretreatment Figure 22. Effect of cAMP analog on EGF-induced PGEj production Two days after plating, WISH cells were preincubated for 4 hr with or without 5 x 10'5 M dibutyryl cAMP (Db-cAMP) in F12-DMEM/NCS medium. Thereafter, cells were rinsed once with serum-free F12/DMEM medium and then exposed to F12/DMEM/NCS medium in the presence or absence of 10 ng/ml EGF for an additional 4 hr. The concentration of PGE2 in the medium was determined by RIA. The data represent the mean ± SEM of six replicates per condition. **: p < 0.001 compared with EGF treatment only. 68 6 Posttreatment: c . f~| Control • rH QJ 5 ^ e g f 10 ng/ml □ c_ CL A CD 3 CD c 2 cu LU CD CL 1 0 Control Forskolin Pretreatment Figure 23. Effect of an adenylate cydase activator on EGF-induced PGEjj production Two days after plating, WISH cells were preincubated for 4 hr with or without 5 x 10* M forskolin in F12/DMEM/NCS medium. Thereafter, cells were rinsed once with serum-free F12/DMEM medium and then exposed to F12/DMEM/NCS medium in the presence or absence of 10 ng/ml EGF for an additional 4 hr. The concentration of PGE2 in the medium was determined by RIA. The data represent the mean ± SEM of six replicates per condition. *: p<0.05 compared with EGF treatment only. 69 4 Posttreatment: |~1Control a • r - i ^ egf 10 ng/ml CD 799249 a-> O c_ 3 Q. c n £ 2 c n c OJ LU 1 CD Q_ 0 Control IBMX Pretreatment Figure 24. Effect of a phosphodiesterase inhibitor on EGF-induced PGEj production Two days after plating, WISH cells were preincubated for 4 hr with or without 5 x 10 s M IBMX in F12/DMEM/NCS medium. Thereafter, cells were rinsed once with serum-free F12/DMEM medium and then exposed to F12/DMEM/NCS medium in the presence or absence of 10 ng/ml EGF for an additional 4 hr. The concentration of PGE2 in the medium was determined by RIA. The data represent the mean ± SEM of six replicates per condition. **: p< 0.001 compared with EGF treatment only. 70 25 cz • f—H CD - t - > 20 O c_ CL c n 15 o £ 10 CL 5 <=c C_) 0 0 1 2 3 4 Time (hr) Figure 25. Time-dependent effect of forskolin on cAMP accumulation in WISH cells Two days after plating, WISH cells were incubated with 1 0 M IBMX in the presence (closed triangle) or absence (open triangle) of 5 x 10*6 M forskolin in F10/HEPES/BSA medium. At the indicated time, 2 ml of 10 % TCA was added to the well to stop cAMP metabolism. Cyclic AMP concentration was determined by RIA. The data represent the mean ± SEM of six replicates per condition. 71 15 QJ - t - J O c_ CL 10 c n o e □L 5 0 Control q 20 40 60 Forskolin (^M) Figure 26. Dose-dependent effect of forskolin on cAMP accumulation in WISH cells Two days after plating, WISH cells were incubated with 1 0 A M IBMX and the indicated concentrations of forskolin in F10/HEPES/BSA medium. After a 5-min incubation, 10 % TCA was added to the plates to stop cAMP metabolism. Cyclic AMP concentration was determined by RIA. The data represent the mean ± SEM of six replicates per condition. Control: cells received IBMX only. 72 4 CD - * - > 3 O c_ o . CD 2 CD d ru LU 1 CD CL 0 Control EGF 9 -8 -7 -6 -5 -4 treated Log [Forskolin] (M) in pretreatment Figure 27. Dose-dependent effect of forskolin on EGF-induced PGE2 production Two days after plating, WISH cells were preincubated for 4 hr with the indicated concentrations of forskolin in F12/DMEM/NCS medium. Thereafter, the cells were rinsed once with serum-free F12/DMEM medium and then exposed to F12/DMEM/NCS medium in the presence (closed triangle) or absence (open triangle) of 10 ng/ml EGF for an additional 4 hr. The concentration of PGE2 in the medium was determined by RIA. The data are presented as mean ± SEM of six replicates per condition. (Control: cells received medium only at both treatment phases; EGF-treated: cells received medium at the pretreatment phase and 10 ng/ml EGF at the posttreatment phase. *: p<0.05, **: p < 0.001, ***: p < 0.0001 compared w ith EGF treatment only.) 73 Effect of terbutaline and ritodrine on EGF-induced PGE2 production To assess whether tocolytic drugs can inhibit EGF-induced PGE2 production, WISH cells were preexposed to either terbutaline or ritodrine, two widely used tocolytics, for 4 hr prior to exposure to EGF. As shown in Fig. 28, exposure of WISH cells to concentrations of 10"8 to Iff4 M of terbutaline alone had little effect on PGE2 production; however, EGF- induced PGE2 production was inhibited by prior to exposure of the cells to terbutaline. Terbutaline at 10'7, 10*6, 10 s, and lO4 M reduced EGF-induced PGE2 production by 16.2% (p = 0.0317), 27.1% (p = 0.0108), 31.6% (p = 0.0002) and 31.4% (p=0.0003), respectively. Similarly, ritodrine at a concentration of 10 s M reduced EGF-induced PGE2 production by approximately 20.6% (Fig. 29). These results are in accord with the observation that terbutaline is a more potent tocolytic agent than ritodrine. Role of cAMP in the inhibitory effect of terbutaline and ritodrine on EGF-induced PGE2 production It has been suggested that the tocolytic effects of terbutaline and ritodrine are due to their ability to increase intracellular cAMP, which phosphorylates myosin light-chain kinase, resulting in a decreased ability of the uterine muscle to contract [111. Their effect on other tissues related to parturition has not been established. Therefore, experiments were designed to test whether terbutaline and ritodrine stimulate cAMP 74 5 CD -4-J 4 O c_ CL CD 3 CD C 2 OJ LU CD D_ 0 Control EGF 8 -7 -6 -5 -4 Log (Terbutaline) (H) in pretreatment Figure 28. Dose-dependent effect of terbutaline on EGF-induced PGE2 production Two days after plating, WISH cells were preincubated for 4 hr with the indicated concentrations of terbutaline in F12/DMEM/NCS medium. Thereafter, the cells were rinsed once with serum-free F12/DMEM medium and exposed to F12/DMEM/NCS medium in the presence (closed triangle) or absence (open triangle) of 10 ng/ml EGF for an additional 4 hr. The concentration of PGE2 in the medium was determined by RIA. The data are presented as mean ± SEM of six replicates per condition. (Control: cells received medium only at both treatment phases; EGF-treated: cells received medium at the pretreatment phase and 10 ng/ml EGF at the posttreatment phase. *: p<0.05, **: p < 0.001 compared with EGF treatment only.) 75 Posttreatment: cz 5 flcontrol • r—1 CD -*—> ^EGF 10 ng/ml o 4 c_ Q. c n E 3 c n 2 OJ LU CD Q_ 1 0 Control Ritodrine Pretreatment Figure 29. Effect of ritodrine on EGF-induced PGE2 production Two days after plating, WISH cells were preincubated for 4 hr with or without 5 x 10 s M ritodrine in F12-DMEM/NCS medium. Thereafter, cells were rinsed once with serum-free F12/DMEM medium and exposed to F12/DMEM/NCS medium in the presence or absence of 10 ng/ml EGF for an additional 4 hr. The concentration of PGE2 in the medium was determined by RIA. The data represent the mean ± SEM of six replicates per condition. (*: p<0.05 compared with EGF treatment only.) 76 accumulation in WISH cells, and whether the inhibitory effect of these two tocolytics on EGF-induced PGE2 production is mediated by cAMP. To examine whether these tocolytic drugs stimulate cAMP accumulation, WISH cells were exposed to either 10 s M of terbutaline or ritodrine for times from 30 seconds to 30 min. As shown in Fig. 30 and Fig. 31, both terbutaline and ritodrine caused a time-dependent increase in cAMP. WISH cells were next exposed to either terbutaline or ritodrine at concentrations ranging from 10'9 to lO-4 M for 5 min. The results shown in Fig. 32 indicate that terbutaline stimulated cAMP accumulation in a dose- dependent manner. The maximal effect occurred at concentrations higher than 104 M. Similarly, ritodrine caused a dose-dependent but less effective increase in WISH cell cAMP (Fig. 33). On the basis of these experiments, it is clear that terbutaline and ritodrine can stimulate cAMP in WISH cells. To determine whether the effect of terbutaline and ritodrine on EGF- induced PGE2 production results from their effects on cAMP, WISH cells were exposed to the /3-blocker butoxamine prior to treatment with tocolytic agents and EGF. As expected, terbutaline- and ritodrine-inhibited EGF responses were blocked by 10 '5 M of butoxamine (Fig. 34 and Fig. 35). On the basis of these results, it is concluded that /3-adrenergic tocolytic agents also alter EGF-induced PGE2 production and this effect is via a /32-adrenoceptor-coupled adenylate cyclase. 77 5 CD • 4 - J O 4 c_ CL CD 3 O e CL 2 .A — 1 CD 0 0 5 Time (min) Figure 30. Time-dependent effect of terbutaline on cAMP accumulation in WISH cells Two days after plating, WISH cells were incubated with 10^ M IBMX in the presence (closed triangle) or absence (open triangle) of 10'5 M terbutaline in F10/HEPES/BSA medium. At the indicated time, 2 ml of 10 % TCA was added to the well to stop cAMP metabolism. Cyclic AMP concentration was determined by RIA. The data represent the mean ± SEM of six replicates per condition. 78 c z 3 • r ~ ~ i CD -M O c_ CL CD 2 E O i z ; e CL 1 -SC C_) 0 0 5 10 15 20 25 30 Time (min) Figure 31. Time-dependent effect of ritodrine on cAMP accumulation in WISH cells Two days after plating, WISH cells were incubated with 10"4 M IBMX in the presence (closed triangle) or absence (open triangle) of 10 s M ritodrine in F10/HEPES/BSA medium. At the indicated time, 2 ml of 10 % TCA was added to the well to stop cAMP metabolism. Cyclic AMP concentration was determined by RIA. The data represent the mean ± SEM of six replicates per condition. 79 4 c • «—I CD ■±~> 3 O c_ CL c n 2 o e CL C_) 0 Control 9 8 7 6 5 4 Log [Terbutaline] (M) Figure 32. Dose-dependent effect of terbutaline on cAMP accumulation in WISH cells Two days after plating, WISH cells were incubated with lO"4 M IBMX and the indicated concentrations of terbutaline in F10/HEPES/BSA medium. After a 5-min incubation, 10 % TCA was added to the plates to stop cAMP metabolism. Cyclic AMP concentration was determined by RIA. The data represent the mean ± SEM of six replicates per condition. (Control: cells received IBMX only; *: p<0.05, ***: p < 0.0001 compared with control.) 80 4 cz • r i QJ -*-> O 3 C_ CL c n 2 o e CL Q- 1 0 Control ■9 -8 -7 -6 -5 -A Log [Ritodrine] (M) Figure 33. Dose-dependent effect of ritodrine on cAMP accumulation in WISH cells Two days after plating, WISH cells were incubated with 10"4 M IBMX and the indicated concentrations of ritodrine in F10/HEPES/BSA medium. After a 5-min incubation, 10 % TCA was added to the plates to stop cAMP metabolism. Cyclic AMP concentration was determined by RIA. The data represent the mean ± SEM of six replicates per condition. (Control: cells received IBMX only; *: p<0.05, **: p< 0.001 compared with control.) 81 •H 5 • r—f CD A o H c_ CL c n 3 e CD J= 2 C\J lu , CD I Q_ 0 c T B B+T Pretreatment Figure 34. Effect of butoxamine on terbutaline-induced decrease in PGE2 production in response to EGF Two days after plating, WISH cells were preincubated for 30 min with or without 10 s M butoxamine in F12/DMEM/NCS medium. Thereafter, 10 ‘5 M epinephrine was added to some wells for 3.5 hr. After the total 4-hr preincubation, cells were rinsed once with serum-free F12/DMEM medium and then exposed to 10 ng/ml EGF in F12/DMEM/NCS medium for an additional 4 hr. The concentration of PGE2 in the medium was determined by RIA. The data represent the mean ± SEM of six replicates per condition. (C: control; B: butoxamine; T: terbutaline; B+T: butoxamine and terbutaline. All four groups received 10 ng/ml EGF in posttreatment. *: p<0.05 compared with control.) 82 5 CD CL c n 3 c n 3 2 Lu -i CD 1 Q_ 0 c R B B+R Pretreatment Figure 35. Effect of butoxamine on ritodxine-induced decrease in PGE 2 production in response to EGF Two days after plating, WISH cells were preincubated for 30 min with or without 10' 5 M butoxamine in F12/DMEM/NCS medium. Thereafter, 10 s M epinephrine was added to some wells for 3.5 hr. After the total 4-hr preincubation, cells were rinsed once with serum-free F12/DMEM medium and then exposed to 10 ng/ml EGF in F12/DMEM/10% NCS medium for an additional 4 hr. The concentration of PGE2 in the medium was determined by RIA. The data represent the mean ± SEM of six replicates per condition. (C: control; B: butoxamine; R: ritodrine; B + R: butoxamine and ritodrine. All four groups received 10 ng/ml EGF in posttreatment. *: p< 0.05 83 VI. Relationship between cAMP and PGE2 in WISH cells From previous experiments, it is clear that the effects of catecholamines on EGF-induced PGE2 production are due to an increase in cAMP. When the effects of several different doses of epinephrine and terbutaline on cAMP are plotted against the effects of the same doses on EGF-induced PGE2 production, the direct relationship between the ability to increase cAMP and decrease PGE2 is apparent (Fig. 36). Although cAMP has been shown to influence PG synthesis in several types of cells [104,105], the data shown in this dissertation indicate that none of the tested cAMP-stimulating agents directly stimulated or inhibited PGE 2 production in WISH cells. Since there is evidence that PGE2 can stimulate cAMP production in macrophages [106], the effect of PGE2 on cAMP accumulation in WISH cells was determined. As shown in Fig. 37, PGE2 caused a time-dependent effect on cAMP accumulation, which peaked at 30 seconds after PGE2 exposure. In addition, PGE2-stimulated cAMP accumulation was dose-dependent, reaching a maximum at doses higher than 250 ng/ml (Fig. 38). On the basis of the experiments, the results show, for the first time, that PGE2 stimulates cAMP accumulation in WISH cells. Since EGF has been shown to influence PGE 2 production as well as cAMP accumulation [108], the interaction betw een EGF, PGE 2 and cAMP in WISH cells was also evaluated. The results shown in Fig. 39 indicate that EGF had no effect on cAMP accumulation either in the presence or absence Figure 36. Correlation, betw een the effects of cAM P-stimulating agents agents P-stimulating cAM of effects the een betw Correlation, 36. Figure % decrease in EGF-induced Q_ CD LU ■o . o c O CJ 13 o _ ru in WISH cells WISH in ncM cuuainado G-nue PGE EGF-induced on and accumulation cAMP on 20 50 30 40 0 1 0 080 50 ices i cAMP accumulation in increase % A Terbutaline A = 0.9763 r= A Epinephrine 110 140 2 production production 170 84 85 8 cz • rH CD - i- > O 6 c_ Q. CD 4 O s CL □_ 2 «=c u 0 0 10 20 30 Time (min) Figure 37. Time-dependent effect of PGE2 on cAMP accumulation in WISH cells Two days after plating, WISH cells were incubated with 10*1 MIBM X in the presence (closed triangle) or absence (open triangle) of 250 ng/ml PGE2 in F10/HEPES/BSA medium. At the indicated time, 2 ml of 10 % TCA was added to the well to stop cAMP metabolism. Cyclic AMP concentration was determined by RIA. The data represent the mean ± SEM of six replicates per condition. 86 CD 4 ■ * -> O c_ Q. 3 cn o £ 2 o . Q_ s : 1 0 0 500 1000 1500 2000 PGE2 (ng/ml) Figure 38. Dose-dependent effect of PGE2 on cAMP accumulation in W ISH cells Two days after plating, WISH cells were incubated with 10A M IB MX and the indicated concentrations of PGE2 in F10/HEPES/BSA medium. After a 5-min incubation, 10 % TCA was added to the plates to stop cAMP metabolism. Cyclic AMP concentration was determined by RIA. The data represent the mean ± SEM of six replicates per condition. 87 cz Ql ------C Epi EGF Epi+EGF Treatment Figure 39. Effect of EGF on epinephrine-stimulated cAMP accumulation in WISH cells Two days after plating, WISH cells were incubated with 5 x 10 ' 5 M IBMX and with either 10 M epinephrine, 10 ng/ml EGF or both in F10/HEPES/BSA medium. After a 5-min incubation, 10 % TC?A was added to the plates to stop cAMP metabolism. Cyclic AMP concentration was determined by RIA. The data represent the mean ± SEM of six replicates per condition. 88 of epinephrine. As shown in Table 3, neither 10 s M epinephrine nor KT1 M dibutyryl cAMP stimulated PGE2 production, and neither one enhanced EGF-induced PGE2 production. Since catecholamines alone had no effect on PGE2 production, but did decrease EGF-induced PGE2 production, it may be concluded that the effect on PGE2 production is probably mediated by cAMP. In addition, since EGF alone had no effect on cAMP accumulation, the increase in cAMP is not mediated by EGF. VII. Effect of catecholamines on EGF radioligand binding Since binding of EGF to its receptor is the initial step in transduction of the EGF signal, regulation of the number of receptor sites or their affinity for EGF by catecholamines might be responsible for the observed effect of catecholamines on EGF induced PGE2 production by WISH cells. A series of experiments was designed to test this possibility. All binding studies were conducted at 4 °C to prevent receptor internalization. Kinetics of l12SI]EGF binding to WISH cells Binding studies were first conducted to establish the optimal time required to reach equilibrium binding in WISH cells. Specific binding of EGF was determined by incubating cells with 50 pM of [ 125 I]EGF in the presence or absence of 250 ng/ml of unlabeled EGF, for times from 15 min to 6 hr. As shown in Fig. 40, I125UEGF binding increased with the time of 89 Table 3. Lack of effects of epinephrine and dibutyxyl cAMP on EGF-induced PGE2 production by WISH cells Two days after plating, WISH cells were incubated for 4 hr with the test substances in F12/DMEM/NCS medium. The concentration of PGE 2 in the medium was determined by RIA. The data represent mean ± SEM of six replicates per condition. Treatm ent______PGE2 (ng/mg protein) Control 0.50 ± 0.03 Epinephrine (10‘5 M) 0.47 ± 0.02 Dibutyryl cAMP (1 0 -4 M) 0.42 ± 0.16 EGF (10 ng/ml) 1.63 ± 0.11 Epinephrine + EGF 1 .6 6 ± 0.06 Dibutyryl cAMP + EGF 1.54 ± 0.07 90 CD c • H TD a • i— i cz jQ • r —4 CD CJ j- J O C_ CL CJ CD CD CL tn CD LU in C\J 0 0 1 2 3 4 5 6 Time (hr) Figure 40. Kinetics of [mI]EGF binding to WISH cells at 4°C WISH cells in 12-well plates were incubated with 50 pM of I^IJEGF for the indicated time as described in Methods. Specific binding was the difference between binding in the presence or absence of 250 ng/ml of unlabelled EGF. Each point is the mean of triplicate samples. The data were plotted using the computer program Inplot (rectangular hyperbola). 91 incubation, and reached saturation at approximately 4 hr. Competitive binding studies In order to determine the concentration required to compete with 50 % of EGF binding (ICS0), cells were incubated with 50 pM [ 125 I1EGF in the presence of increasing concentrations of unlabeled EGF (0-250 ng/ml) for 4 hr at 4 °C. The results indicate that the concentration of unlabeled EGF which inhibited 50 % of the binding was approximately 0.5 ng/ml (Fig. 41). Experiments were next conducted to test whether catecholamine pretreatment alters the competitive binding. Since results from our laboratory and others [1091 show that binding of [ 125 I]EGF to WISH cells can be reduced by phorbol ester, phorbol 12,13-myristate acetate (PMA) was used as a positive control in the EGF binding assay. WISH cells were pretreated with either 10'5 M epinephrine or 10'7 M PMA for 4 hr at 4 °C, and then incubated with 50 pM of [ 12SI]EGF in the presence or absence of 0-250 ng/ml of unlabeled EGF. As expected, EGF binding was reduced in cells pretreated with PMA. However, epinephrine pretreatment did not change the competition curve compared with control (Fig. 42). A further examination of the effect of epinephrine on binding indicated that [12SIJEGF specific binding was not altered by preexposure of WISH cells to epinephrine at concentrations ranging from 1(T7 M to 10"4 M, and was only • reduced approximately 15% by epinephrine at a very high dose, 10'3 M (Fig. 92 CD 100 c H r—H o c_ c o u o .01 1 1 10 100 1000 EGF (ng/ml) Figure 41. Inhibition of [mI]EGF binding to WISH cells by EGF WISH cells in 12-well plates were incubated with 50 pM [mI]EGF in the presence of various concentrations of unlabeled EGF for 4 hr. The data are expressed as the percentage of control, which is the binding in the absence of unlabeled EGF. Each point is the mean of triplicate samples. The data were plotted using the computer program Inplot (sigmoid curve). 93 100 CD a -o cr o c_ A - > c z o CJ o .01 .1 1 10 100 1000 EGF (ng/ml) Figure 42. Effects of epinephrine and PMA on I^IJEGF competitive binding WISH cells in 12-well plates were pretreated with either medium (open square), 10'5 M epinephrine (closed triangle) or 10'7 M PMA (closed diamond) for 4 hr at 4 °C, and then incubated with 50 pM of [^IJEGF in the presence of various concentrations of unlabeled EGF for 4 hr. The data are expressed as the percentage of control, which is the binding in the absence of unlabeled EGF. Each point is the mean of triplicate samples. concentrations of epinephrine for 4 hr at 37 °C prior to EGF binding assay control.) assay ith w binding compared EGF to prior p<0.05 °C (*: 37 at hr 4 Methods. for in described epinephrine as of binding concentrations specific IlEGF [m on epinephrine of Effect 43. Figure Bound (f mol/mg’ protein) WISH cells in 12-well plates were incubated w ith various various ith w incubated were plates 12-well in cells WISH 20 80 40 60 0 Control 7 o [pnprn] (M) [Epinephrine] Log -5 6 4 3 94 95 43). On the basis of the results, it is concluded that the inhibitory effect of epinephrine on PGE2 production induced by EGF may be not due to an alteration in the ability of WISH cells to bind EGF. Saturation binding isotherms In addition to competition studies, saturation binding isotherms were conducted to quantitate the apparent affinity (Kd) and binding site concentration (Bmax) of the EGF receptor in WISH cells. A preliminary study showed that the specific binding of [125 I]EGF increases with increasing concentration of radiolabeled ligand and reaches saturation at 1-2 nM (data not shown). When WISH cells were incubated with increasing concentrations of [125 I]EGF ranging from 0.001-0.8 nM (Figure 44), the [125 I]EGF specific binding increased with increasing concentrations of labeled ligand. When the data was plotted using a rectangular hyperbola equation to determine the line of best fit (Fig. 45), the calculated Kd was 0.27 ± 0.03 nM and 141.1 ± 6.2 fmol/mg protein for Kd and Bmax, respectively. Subsequent experiments were designed to examine the effect of epinephrine pretreatment on [125 I]EGF specific binding. WISH cells were pretreated with or without 10 s M epinephrine for 4 hr prior to 125 [ I]EGF exposure. The results shown in Table 4 are the values of the mean ± SEM of Kd and Bmax from four separate experiments with and without epinephrine. Analysis by Student's t-test indicates that there is no significant change in Kd, but there 96 120 100 ■o a Z) CD o X3 o CD. CD A Total binding cn • Specific binding ANon-specific binding in CM o 0 A ,8 Total [1 2 5 I] EGF added (nM) Figure 44. Saturation binding isotherm of I^IJEGF binding to WISH cells WISH cells in 24-well plates were incubated with 0.001 to 0.8 nM of [^IJEGF in the presence (open triangle) or absence (closed triangle) of 250 ng/ml unlabeled EGF for 4 hr. Specific binding was the difference between binding in the presence or absence of unlabelled EGF. 97 CJ) cz 120 100 jn c: C_) CD O c_ (_j CL CL) CD CL CO e Kd= 0.27 t 0.03 (nM) Bmax' 141.1 » 6.2 (fmol/mg protein) CD o in CVJ 0 .2 A .6 .8 Total [125I]EGF added (nM) Figure 45. Rectangular hyperbola plot of [125 I]EGF specific binding to WISH cells The figure 45 was replotted in a rectangular hyperbola, and KD and Bmax were calculated by the computer. 98 Table 4. Saturation binding isotherm of [^IJEGF binding to WISH cells pretreated with epinephrine WISH cells in 24-well plates were pretreated with 10 s M epinephrine for 4 hr prior to conducting the saturation binding isotherm as described in Methods. The data represents the mean ± SEM of Kd (apparent affinity) and Bmax (binding site concentration) of the EGF receptor from four separate experiments for each condition (*: p<0.05). Kd (nM)______B__ (fmol/mg protein) Control 1.28 + 0.48 177.2 ± 28.1 Epinephrine 0.52 ± 0.18 * 98.1 ± 12.9 99 is a significant decrease in the Bmax of epinephrine-treated cells. On the basis of the results, it is concluded that the inhibitory effect of epinephrine on EGF-induced PGE2 production may be mediated by changes in the Bmax of the EGF receptor. VIII. Incorporation of arachidonic add into phospholipids Arachidonic acid is the precursor of PGE2, and an increase in PGE2 production by the cell may result from a change in the availability of arachidonic acid. It has been shown that the control of arachidonic acid mobilization and subsequent prostaglandin production is an important regulatory process for the initiation and maintenance of labor. Human amnion contains the enzymatic activity to release arachidonic acid from phospholipids, and a significant amount of arachidonic acid is lost from phosphatidylethanolamine and phosphatidylinositol with the onset of labor 150]. However, little is known of the effect of labor-inducing factors on the arachidonic acid system in amnion cells m vitro. For this reason, experiments were designed to study the incorporation of arachidonic acid into WISH cell lipids to determine (1) the proportional amount incorporated into the major phospholipid species, and 2 ) ( whether the amount of incorporation varies with EGF treatment. In order to determine the time required to achieve isotopic steady state in WISH cells, cells were incubated in F12/DMEM/0.5% FAF-BSA containing 100 1.5 f i d of [3HJarachidonic acid for the indicated time periods. As shown in Fig. 46, a labeling period of 12 hr was required to achieve isotopic steady state in WISH cells. To determine the proportional amount incorporated into the major glycerophospholipid species, WISH cells were labeled with 2 jtCi of 3H][ arachidonic acid for 1 2 hr, and cell membrane lipids were extracted and separated by TLC. In a solvent system of chloroform /ethanol /water/ triethylamine (30:34:8:35,v/v), four peaks were revealed. The results indicate that the major glycerophospholipids in the WISH cell membrane are phosphatidylcholine 2 ( 0 . 2 %), phosphatidylserine (18.9%), phosphatidylinositol (25.5%) and phosphatidylethanolamine (35.4%) (Fig. 47). To determine whether the amount of incorporation varies with EGF treatment, WISH cells were labeled with 2 fiC i of [3H]arachidonic acid in the presence or absence of 10 ng/m l EGF. The results (Fig. 48) indicate that EGF increased the total phospholipid incorporation, especially increasing the incorporation of arachidonic acid into phosphatidylserine and phosphatidylethanolamine. Since EGF induces changes in the incorporation of arachidonic acid into WISH cells, and epinephrine can inhibit the effect of EGF on PGE2 production, experiments were designed to determine whether the epinephrine-induced decrease in PGE2 production in response to EGF could 101 3 Q_ Q CD I 2 O CO 1 4-> cz Z) o CJ) 0 0 6 12 18 2 A Time (hr) Figure 46. Time-dependent effect of [3H]arachidonic add incorporation into total phospholipids in WISH cells WISH cells in 60 mm-dishes were incubated with 1.5 f i d of [3H] arachidonic acid for times from 1 to 24 hr. At the indicated time, cells were scraped from dishes and phospholipids were extracted as described in Methods. Each point represents the mean of two samples. 102 III 100 o X 40- c =3 O CJ AAA*A AAAAAAA 0 5 10 15 20 25 30 35 Fraction number Figure 47. Incorporation of 3[H]arachidonic add into WISH cell phospholipids WISH cells in 60 mm-dishes were incubated with 2 / i d of I3HJarachidonic acid for 12 hr. Phospholipids were extracted as described in Methods. Four peaks were revealed by TLC. (I: phosphatidylcholine, Rf= 0.16; II: phosphatidylserine, R*= 0.25; III: phosphatidylinositol, R(= 0.28; and IV: phosphatidylethanolamine, Rf= 0.32.) 103 150 Q_ CD 100 o X a 3 O C_D A'A 0 5 10 15 20 25 30 35 Fraction number Figure 48. Effect of EGF on [3HJarachidonic acid incorporation into WISH cell phospholipids WISH cells in 60 mm-dishes were incubated with 2 f i d of [3H]arachidonic acid in the presence (closed triangle) or absence (open triangle) of 10 ng/ml EGF. After 12 hr incubation, phospholipids were extracted as described in Methods. 104 be explained by changes in the mobilization or utilization of the phospholipids. WISH cells were labeled with 2n d of [3Hlarachidonic acid for 24 hr, and cells were then treated w ith or w ithout 10'5 M epinephrine for 4 hr. As shown in Table 5, there are no significant changes in any of the four phospholipids in cells treated with epinephrine. This suggests that epinephrine does not induce a change in the mobilization or utilization of the phospholipids. On the basis of these experiments, it is concluded that EGF increases incorporation of arachidonic acid into phospholipids in WISH cells, and that preexposure of WISH cells to epinephrine may not change the mobilization or utilization of the phospholipids for PGE2 production in response to EGF. IX. The activity of PGH 2 synthase in WISH cells Recent studies 171] have shown that EGF regulates PG production by induction of PGH2 synthase. If induction of PGH2 synthase is a critical step in EGF-induced PG production, then it is reasonable to assume that catecholamines may modulate EGF action in WISH cells by altering the ability of EGF to induce this enzyme. To assess the activity of PGH 2 synthase, the rate of conversion of [3H]arachidonic acid to [3H]PGE2 was determined in sonicated preparations of treated WISH cells. The first experiment was designed to determine whether either epinephrine or EGF alone affects the PGH2 synthase activity. As shown in 105 Table 5. Effect of epinephrine on [3H]arachidonic add incorporation into phospholipids in response to EGF WISH cells in 60 mm-dishes were incubated with 2 f i d of [3H]arachidonic acid for 24 hr. The cells were then pretreated with or without 10'5 M epinephrine. After a 4-hr preincubation, cells were exposed to 10 ng/ml EGF for an additional 4 hr. Phospholipids were extracted as described in Methods. Phospholipid % of control Phosphatidylcholine 102.4 Phosphatidylserine 94.8 Phosphatidylinositol 105.2 Phosphatidylethanolam ine 113.6 106 Fig. 49, treatment of WISH cells with 10'5 M epinephrine for 4 hr did not significantly change the enzyme activity. In contrast, the activity of PGH2 synthase increased by 3-fold in EGF-treated cells. This is in accord with the results of Casey et al. [711, who found that PGH2 synthase was increased by 2-5 fold in cells treated with EGF. To examine whether preexposure of WISH cells to epinephrine alters EGF-induced PGH2 synthase activity, cells were incubated with or without 10'5 M epinephrine for 4 hr prior to EGF exposure. The results indicate that there was no significant change in the enzyme activity in cells pretreated with epinephrine compared with that of control cells (Fig. 50). On the basis of these experiments, it is concluded that EGF by itself has the ability to induce PGH2 synthase activity; however, epinephrine-pretreatment has no effect on the EGF-induced PGH2 synthase activity. Therefore, the inhibitory effect of epinephrine on EGF-induced PGE2 production is not due to the alteration of the activity of PGH2 synthase. 107 CD -i—> O c_ ■ o CL CD e CD C_ o (M UJ CD D_ O e CL Control Epinephrine Treatment Figure 49. Effects of epinephrine and EGF on PGH2 synthase activity WISH cells were incubated with either 10 s M epinephrine or 10 ng/ml EGF for 4 hr. Thereafter, the cells were scraped from the dished; preparations of cells and assays were conducted as described in Methods. Data are the mean ± SEM of three samples. 108 cr ■ i — i 4-»CD o c_ •o> o o . £ c_ cn o 9999999999 CM LU CD D. o e CL Control Epinephrine Pretreatment Figure 50. Effect of epinephrine on EGF-induced PGH2 synthase activity WISH cells were pretreated with or without 10 s M epinephrine for 4 hr prior to EGF exposure. Thereafter, the cells were scraped from the dishes; preparations of cells and assays were conducted as described in methods. Data are the mean ± SEM of three samples. DISCUSSION I. Use of human amnion-derived WISH cells as a model system to study the regulation of PGE2 biosynthesis in parturition The endocrine or paracrine factors responsible for the initiation of human parturition remain unknown; however, it is likely that PGs play a primary role. One hypothesis suggests that the communication between the fetus and the mother may occur by way of the action of fetal secretory products (labor-initiating signals) that enter the amniotic fluid, acting on the fetal membranes to produce PGs [25,26]. In particular, the amnion has been proposed to be a target tissue for labor-initiating signals emanating from the human fetus [55,110] and is considered to be a major source of PGs (particularly PGE2) produced by intrauterine tissue at term [1,112]. Based on morphology, lipid composition and enzymatic activities, amnion cells in primary culture are an appropriate in vitro model system for investigating the regulation of PG biosynthesis [113,114]. While primary amnion cell cultures are very useful and have been used by several laboratories, different culture preparations may vary considerably. For example, it has been reported that EGF acts to stimulate PGE2 production 109 110 over a w ide range (2-150 fold) in hum an amnion primary cell cultures [71]. In addition, there are difficulties in undertaking studies with cells in primary culture, such as tissue availability and low cell yields. Thus, in this dissertation, a human amnion cell line (WISH) was used as the model system to investigate PGE2 production. The WISH cell line provides a more homogenous and more stable population than do primary amnion cells. Several physiologically important substances, such as growth factors and immunoregulatory agents, stimulate PGE2 production in WISH cells in a manner similar to that observed in primary cultures [94]. Previous studies from our laboratory and others [94,115] suggest that WISH cells are an effective model for the study of the regulation of PG biosynthesis in amnion. U. Role of EGF and catecholamines in human parturition EGF, which can be produced by the fetal kidney [32], may be the critical labor-inducing signal from the fetus. Several lines of evidence support a role of EGF as aprimary inducer of human parturition. For example, (1) The concentrations of EGF in amniotic fluid rise with increased gestational age [70]; (2) The median concentration of EGF in non-laboring women was found to be 1.28 ng/ml, while the median for laboring women increased to 5 ng/m l [70]; (3) Hum an am nion contains EGF receptors [71]; (4) EGF stimulates PGE2 production by amnion cells [72]; and (5) EGF induces I l l uterine contractions in an in vitro system [116]. In addition to EGF, other fetal products, such as PAF, IL-1 and TNF, have been found in amniotic fluid. While these fetal products can stimulate PGE2 production by amnion cells, none has been shown to increase as a funation of gestational age. In fact, the levels of PAF, IL-1 and TNF in amniotic fluid increase in infection and they have been suggested to play a role in infection-associated preterm labor [2]. Since the levels of EGF in amniotic fluid do not increase in infection [70], EGF alone dose not seem to play a major role in infection- associated preterm labor, but it is thought to play as a primary inducer of parturition at term. It has been reported that there are increases in the concentration of catecholamines in amniotic fluid [101,102] and increases in the number of /3-adrenergic receptors in human amnion tissue during late pregnancy [103]. These /3-adrenergic receptors are functional, since cAMP accumulation in amnion cells can be stimulated with catecholamines. The increase in cAMP could be blocked by the /3-blocker propranolol but not by the a-blocker phentolamine, implying action through /3-adrenergic receptors on the amnion. Thus, it seems that catecholamines in amniotic fluid may play a role in human parturition. The results presented in this dissertation show a decrease in EGF-induced PGE2 production after exposure of WISH cells to catecholamines. Therefore, this dissertation proposes a central role of EGF and a regulatory role of catecholamines in the initiation of human labor. 112 III. Relationship between cAMP and PGE2 in WISH cells There is evidence that cAMP can either stimulate or inhibit PG synthesis in several types of tissue. For example, Warrick et al [104] showed that cAMP-stimulating agents as well as /3-adrenergic receptor agonists stimulated PG production in dispersed cells from human amnion and decidua, suggesting that cAMP mediates PG synthesis in response to /3- adrenergic receptor stimulation. In contrast, cAMP has been shown to inhibit PG production in bovine vascular endothelial cells [118] and human platelets [1051. The interaction of cAMP and PGs is not unidirectional, since PGs are also known to influence cAMP. For example, PGE2 stimulates cAMP in macrophages [106]. In contrast, PGs inhibit cAMP accumulation in adipose tissue [107]. In human amnion-derived WISH cells, the data shown in this dissertation indicate that cAMP-stimulating agents (i.e., dibutyryl cAMP, IBMX, and forskolin) as well as /3-adrenergic agents (i.e., epinephrine and norepinephrine) do not stimulate PGE2 production. The finding differs from the study by Warrick et al. [104], but it is in accord w ith the study by Casey et al. [93] , which showed that dibutyryl cAMP (10 3 M) and isoproterenol (10's M) fail to trigger PGE2 production in primary human amnion cell culture. The findings shown in this dissertation are also in agreement with the study of Acker et al., [119] which reported that epinephrine at a concentration of 10"6 M has no effect on PGE2 production by amnion cells. 113 This suggests that the observed difference between the finding presented in this dissertation and that of Warrick et al. 1104] is not solely due to the use of the WISH cell line. The results presented in this dissertation show cAMP accumulation in WISH cells during in vitro experiments. Catecholamines stimulate this accumulation, andbutoxamine, a /32-adrenergic receptor antagonist, abolishes the stimulatory effects, suggesting that this stimulation of cAMP accumulation in amnion cells is mediated by /?2-adrenergic receptors. This result is in accord with the study of Di Renzo et al. 1103], which demonstrated that the /3-adrenergic receptors of human amnion were almost entirely /32-subtype. In addition to catecholamines, PGE2 also stimulates cAMP accumulation in WISH cells. Taken together, the results show, for the first time, that cAMP does not stimulate PGE2 synthesis in WISH cells, but PGE2 stimulates cAMP accumulation in this cell system. The PGE2- stimulated cAMP may also inhibit EGF-induced PGE2 production. This implys that WISH cells may have a negative-feed back mechanism to regulate the PGE2 biosynthesis (Fig. 51). Since EGF has been shown to influence both PGE2 production and cAMP accumulation, the effects of EGF on PGE2 production and cAMP accumulation in WISH cells were also evaluated. Some investigators have reported that EGF has an interaction with the adenylate cyclase system. In the human epidermoid carcinoma cell line (A431), EGF by itself does not 114 Catecholamines . EGF I I 3-adrenergic receptor EGF-receptor cAMP PGE; Stimulation < ► Binding * Inhibition ► No effect Figure 51. Regulation of PGE2 production in amnion-derived WISH cells 115 alter the basal level of cAMP. However, it enhances cAMP accumulation in cells treated with cAMP-elevating agents by activating adenylate cyclase [108J. The results in this dissertation indicate that EGF by itself haS no stimulatory effect on cAMP accumulation and does not enhance epinephrine- stimulated cAMP, either. Epinephrine (10'5 M) and dibutyryl cAMP (10"4 M) do not alter the basal level of PGE2, and do not enhance EGF-induced PGE2 production. However, preexposure of amnion cells to catecholamines inhibits EGF-induced PGE2 production although catecholamines alone do not alter PGE2 production. The observation that /3-adrenergic agents can decrease the production of the labor-inducing factor (PGE2) is in accord with previous observations that /3-adrenergic receptor agonists can produce inhibition of uterine activity in the management of preterm labor [12]. These results suggest that in WISH cells, catecholamines stimulate cAMP accumulation, which in turn inhibits PGE2 production in response to EGF. This indicates that the EGF-induced PGE2 biosynthesis pathway is negatively modulated by adenylate cyclase. Since cAMP has been shown to inhibit PLA2 in cultured rat inner m edullary collecting duct cells [120,121] as w ell as other tissues [122], the mechanism by which cAMP inhibits PGE2 production elicited by EGF may be due to interference with liberation of arachidonic acid from cell phospholipids. 116 IV. Role of protein kinase C in EGF-induced PGE2 production Since catecholamines only partially inhibit EGF-induced PGE2 production, it is likely that PGE2 production is influenced by other factors as well. One such factor is protein kinase C, which has been identified and characterized in amnion [123J. Previous studies done in our laboratory have shown that activators of protein kinase C (PKC) augment EGF-induced PGE2 production in WISH cells [115]. Preexposure of WISH cells to 10 9 M PMA alone had little effect on PGE2 production. In contrast, the EGF response was stimulated by prior exposure of cells to PMA. In addition, staurosporine, a PKC inhibitor, decreased EGF-induced PGE2 production at concentration as low as 5 nM. The results imply that PKC activation may be involved in the transduction pathway leading to EGF-induced PGE2 production. Since activation of PKC has been reported to either inhibit [124,125] or stim ulate [126,127] horm one-stim ulated adenylate cyclase, this suggests the possibility of a complex link between adenylate cyclase and PKC in amnion cells. If it is the case that activation of PKC inhibits catecholamine-stimulated cAMP generation, then it suggests a possible role for PKC in "turning off" the catecholamine signals. When EGF-induced PGE2 is regulated by catecholamine via activation of adenylate cyclase, activation of PKC may be also in progress. Therefore, preexposure of WISH cells to catecholamines could not completely inhibit the EGF response. 117 V. Regulation of EGF receptors EGF action on human amnion cells is mediated via the EGF receptor, since an anti-EGF receptor monoclonal antibody was able to block the stimulatory effect of EGF on PGE2 production [731. Many factors have been shown to affect the binding of EGF to certain cells by altering the receptor number and/or affinity. For example, it was reported that tumor necrosis factor (TNF) increased the number of EGF receptors on human fibroblasts [128]. In contrast, platelet-derived growth factor and phorbol esters were shown to decrease the binding of radiolabeled EGF to its receptor [129,130], In the study of Karasaki et al. [109], two subclasses of EGF-binding sites were found on WISH cells (Kdl= 0.16 nM and Kd2= 2.5 nM) by using Scatchard plot analysis [98]. In this dissertation, since the two-site model (double rectangular hyperbola) does not fit the data significantly better than the one-site model (rectangular hyperbola), and since the [12SI]EGF concentrations used were less than 1 nM, the one-site model was used to quantitate the affinity and binding site concentration of the EGF receptor. The results shown in this dissertation suggest that the inhibitory effect of catecholamines on EGF-induced PGE2 production may be mediated by altering the binding sites of the high affinity subclass of the EGF receptor. 118 VI. Regulation of PGH2 synthase activity Arachidonic acid can be converted into PGG2 by the cyclooxygenase reaction, which incorporates two molecules of oxygen into arachidonic acid. The 15-hydroperoxide of PGG2 is then converted to PGH2 by the PG hydroperoxidase reaction. These two enzyme activities have not been resolved in various experimental conditions. It has been suggested that either one enzyme protein contributes to two activities or two separate enzyme activities are tightly bound [1311. Thus, these two enzyme activities are usually referred to as PG endoperoxide synthetase or PGH2 synthase. The study by Casey et al. [71] has shown that EGF stimulates PGE2 production in cultured human amnion cells by a mechanism involved in induction of PGH2 synthase. The results presented in this dissertation also show the stimulatory effect of EGF on the activity of PGH2 synthase in WISH cells. However, preexposure of WISH cells to epinephrine does not change EGF-induced PGH2 synthase activity. Thus, the inhibitory effect of catecholamines on EGF-induced PGE2 is not due to the alteration of the PGH2 synthase activity. VII. Role of cytokines in preterm labor There is evidence that concentrations of PGs in amniotic fluid increase in wom en w ith preterm labor and intraam niotic infection [88,891. In fact, accumulating evidence suggests that there is a strong association between 119 preterm labor and infection [2,82-85]. The mechanisms responsible for the elevated PG levels in preterm labor associated with infection are not well defined. It has been proposed that bacterial products can stimulate maternal and/or fetal cells to produce cytokines (e.g. IL-1, TNF, and PAF), which in turn increase PG biosynthesis by intrauterine tissues and may lead to the onset of labor [2]. An interesting finding in this dissertation is that there is a synergistic interaction between IL-1 and EGF in stimulating PGE2 production by WISH cells (Fig. 9). This observation supports the proposed central role of EGF in the initiation of human labor and may have clinical importance. In cases of preterm labor associated with infection, the increased cytokines may sensitize amnion cells to EGF and thereby amplify EGF-induced PGE2 production. VIII. Conclusions and significance In this in vitro study, there is evidence to support a central role for EGF and a modulating role for catecholamines in the regulation of PGE2 production by human amnion cells. The results suggest that in the human amnion-derived cells (WISH), catecholamines stimulate cAMP accumulation, which, in turn, inhibits PGE2 production in response to EGF. This implies that the pathway of EGF-induced PGE2 biosynthesis is negatively modulated by adenylate cyclase. Previous studies done in our laboratory have shown 120 that EGF-induced PGE2 production can be augmented by PKC activators. The existence of this alternative stimulus for PGE2 production may explain why catecholamines only partially inhibit the EGF effect and suggests the possibility of a complex link between adenylate cyclase and PKC in amnion cells. The data presented in this dissertation indicate that the inhibitory effect of catecholamines on EGF-induced PGE2 production may be due to altering the number of the EGF binding sites. These results also show that epinephrine pretreatment did not change EGF-induced PGH2 synthase activity. This suggests that catecholamine-decreases in EGF-induced PGE2 production may not be due to altering the activity of PGH2 synthase. The significance of this study is that /3-adrenergic agents decrease the production of the labor-inducing factor (PGE2), which is in accord with previous observations that /3-adrenergic receptor agonists are useful inhibitors of uterine activity in the management of preterm labor. In addition, terbutaline and ritodrine (two widely used tocolytics) decrease EGF-induced PGE2 production in human amnion-derived (WISH) cells, suggesting a possible mechanism by which betamimetic drugs produce tocolytic effects and implying not only myometrium but also amnion responses to tocolytics. At present, all of the tocolytic drugs appear to have certain drawbacks and potential adverse side effects. Hence, amnion cells may provide an appropriate system to screen drugs with tocolytic effects. 121 IX. Future studies The expression of the EGF receptor is regulated by receptor synthesis and degradation. The receptor protein can also be regulated at the level of expression of the messenger RNA (mRNA) encoding the protein. For example, rat liver epithelial cells exposed to phorbol esters or epinephrine showed an increase in the expression of EGF receptor mRNA [132]. Thus, it is of interest to examine whether the EGF response inhibited by catecholamines is involved in altering the level of EGF receptor mRNA expression. Boneventre et al. [11] demonstrated that EGF-stimulated PLA2 activity in mesangial cells is a mechanism by which EGF increases PG production. However, Casey et al. [71] reported that EGF acts to stimulate PGE2 production in amnion cells by a mechanism that involved an increase in synthesis of PGH2 synthase without an effect on PLA2 activity. Although EGF-induced PGE2 production in amnion cells is not due to a stimulation of PLA2 activity, it is still possible that the catecholamine-induced decrease in PGE2 production in response to EGF is regulated by the enzyme PLA2. This possibility is supported by evidence that cAMP is able to inhibit PLA2 activity in cultured rat inner medullary collecting duct cells [120] as well as other cells [122,133]. It is now recognized that receptor coupling to adenylate cyclase involves guanine-nucleotide-dependent regulatory proteins (G proteins). Several studies have suggested involvement of G-proteins in 122 receptor-stim ulated PLA2 activity in diverse cell types [134,1351. To get a better understanding of the role of PLA2 in amnion cells, it is of importance to investigate whether cAMP inhibits PLA2 activity and whether there is a G-protein specific for the transduction of receptor-stimulated PLA2 activity. In addition to the cyclooxygenase pathway, arachidonic acid can also be metabolized by the lipoxygenase enzyme in the human amnion, chorion, decidua, and placenta to form hydroxyeicosatetraenoic acids (HETEs) and leukotrienes (LTs) [1361. It has been reported that 12-HETE, 15-HETE, and LTB4 were found in amniotic fluid at term, and that concentrations of all three compounds were higher in amniotic fluid collected from women in labor when compared with women not in labor [1371. 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